selected topics in aptamer research - university of...
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UNIVERSITY OF OTTAWA, FACULTY OF SCIENCE
April 15, 2014
Authored by: Suzanne Elizabeth Kosteniuk, 5282464
Research Supervisor: Dr. Maxim V. Berezovski
Selected Topics in Aptamer Research
Research report submitted in partial fulfillment of the requirements for the course BPS4006
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Abstract
Aptamer research is a young, promising, and highly versatile field. Colloquially referred to as “artificial
antibodies” due to their practical similarities, aptamers are single-stranded oligonucleotides capable of
forming stable three-dimensional structures and binding to targets with high affinity and specificity.
However, aptamers have a number of advantages over antibodies. Aptamers are relatively inexpensive, easy
to synthesize and modify, stable under high temperatures, do not permanently denature, and are versatile
and easy to select. The process through which aptamers for a given target are selected in known as
Systematic Evolution of Ligands by Exponential enrichment (SELEX). SELEX involves repeating rounds of
partitioning a single stranded oligonucleotide pool according its binding to a target, amplifying the group
with the desired binding characteristics, and isolating the amplified single stranded oligonucleotides. The
following report documents three separate projects, each dealing with DNA aptamer research but with
distinctly different focuses and applications.
In the first project, a Polymerase Chain Reaction (PCR) protocol capable of selectively and efficiently
amplifying single stranded DNA oligonucleotides was developed and optimized. The successful
development of this protocol meant that two of the three key steps of SELEX – oligonucleotide
amplification and single stranded oligonucleotide isolation – would be combined into one quick and
efficient step. This project was successful, and the method that was developed was shown to be superior to
Berezovski research group’s previously employed protocol in terms of its efficiency, limit of detection, and
resistance to side-product formation.
The second project dealt with oncolytic viruses, a process known as AptaVIP, and chemical modification of
aptamers. Oncolytic viruses are diverse group of viruses capable of selectively killing cancer cells.
However, their use in therapeutic applications is challenging because when used in vivo they are subject to
degradation mediated by neutralizing antibodies (nAbs). In the past, Berezovski research group has selected
aptamers capable of binding to the oncolytic virus Vesicular Stomatitis Virus ∆51 (VSV∆51) and shielding it
from nAbs-mediated destruction, a concept known as Aptamer-facilitated Virus Protection (AptaVIP).
However, aptamers are subject to in vivo degradation by nucleases, thus aptamer modifications are required
to make AptaVIP feasible from a clinical perspective. In this project, we sought to extend the in vivo half-life
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of these AptaVIP-performing aptamers through conjugation to a 20 kDa polyethylene glycol (PEG moiety).
A method for producing and isolating PEG-modified aptamers was successfully developed and is described
herein. However, the project as a whole was unsuccessful, as it was shown that the aptamers Berezovski
research group previously developed to protect VSV∆51 from nAbs isolated from one antibody-producing
animal are not capable of protecting the virus from nAbs prepared from a different antibody-producing
animal.
In the third and final project, SELEX was employed to select oligonucleotide pools capable of binding to
Vero cells, a common continuous mammalian cell line. Selection of Vero cell-binding aptamers has many
potential applications, including cancer biomarker discovery and enabling novel chemotherapeutic drug
delivery research. Though this project is not yet completed, the oligonucleotide pools that have been
produced in five rounds of SELEX show superior affinity to Vero cells compared to random DNA
oligonucleotide libraries, which is a highly promising result.
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Acknowledgements
First and foremost, I would like to thank my supervisor, Dr. Maxim Berezovski, for giving me the
opportunity to do my honours project in his lab. Dr. Berezovski’s knowledge, generosity, and infectious
enthusiasm for his work has made this an amazing experience for me.
I would also like to thank all of the members of Berezovski lab for making the past year so excellent. I feel
extremely fortunate to have spent the year with such kind, passionate, and wickedly smart people, who are
as comfortable discussing the thermodynamic principles behind oligonucleotide folding as they are debating
whether or not goats are capable of yelling like humans. In particular, I am exceedingly grateful to Darija
Muharemagic, whose mentorship and insight has been absolutely invaluable to me.
I am deeply indebted to my mother, Elizabeth Manning, and my boyfriend, Travis Stewart, for their
support and love throughout this process. They put up with me when my experiments weren’t working and
I was a grumpy jerk, and for that they have my sincerest gratitude.
Finally, I feel I ought to thank Vince Gilligan, George R. R. Martin, and the people of Youtube for
providing me with much needed entertainment for the past year. Without their creativity and talent, I
probably would have finished this project months earlier.
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Table of Contents
ABSTRACT ................................................................................................................................................. I
ACKNOWLEDGEMENTS .............................................................................................................................. III
TABLE OF CONTENTS ................................................................................................................................. IV
INTRODUCTION ........................................................................................................................................ 1
DESIGN OF LINEAR-AFTER-THE-EXPONENTIAL PCR FOR SELEX .................................................................... 3
BACKGROUND ......................................................................................................................... 3
RESULTS AND DISCUSSION ........................................................................................................... 5
CONCLUSIONS ......................................................................................................................... 9
MATERIALS AND METHODS ........................................................................................................ 10
IMPROVING IN VIVO STABILITY OF ONCOLYTIC VIRUS-PROTECTING APTAMERS THROUGH PEGYLATION ........20
BACKGROUND ....................................................................................................................... 20
RESULTS AND DISCUSSION ......................................................................................................... 22
CONCLUSIONS ....................................................................................................................... 26
MATERIALS AND METHODS ........................................................................................................ 27
VERO CELL SELEX ....................................................................................................................................42
BACKGROUND ....................................................................................................................... 42
RESULTS AND DISCUSSION ......................................................................................................... 44
CONCLUSIONS ....................................................................................................................... 45
MATERIALS AND METHODS ........................................................................................................ 46
REFERENCES .............................................................................................................................................50
APPENDIX ...............................................................................................................................................52
FIGURES ............................................................................................................................... 52
PROTOCOL – LATE-PCR FOR SINGLE STRANDED OLIGONUCLEOTIDE POOL AMPLIFICATION ......................... 72
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Selected Topics in Aptamer Research
Introduction
In 1952, James Watson and Francis Crick elucidated the structure of genomic DNA and revolutionized the
fields of chemistry and biology1. Though that discovery occurred scarcely 60 years ago, the technologies
that have been developed in its wake have been astounding. While deoxyribonucleic acid (DNA) and other
nucleic acid molecules in nature serve to transmit genetic information of living organisms, the advent of
technologies to manipulate nucleic acids has led to the use of these molecules in novel, man-made
applications. Aptamers are synthetic single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA)
molecules that form stable three-dimensional structures capable of binding to specific protein, small
molecule, virus, or cell targets, much in the way proteins and small molecules do. The first articles on
aptamer research were published in 1990, shortly after the invention of polymerase chain reaction (PCR) 2,
3, 4.
Aptamer research is still in its infancy, but it is a rapidly growing field with enormous potential. Aptamers
are often compared to antibodies, and can be applied to research and medicine nearly everywhere
antibodies can. The list of current and potential applications for aptamers is substantial. Most notably,
aptamers have applications as therapeutic molecules, like the drug Macugen® for treatment of macular
degeneration, as well as in biosensing, biomarker discovery, biological assays, enhanced drug delivery, and
diagnostic medicine.
Aptamers are discovered via a process known as Systematic Evolution of Ligands by Exponential
Enrichment (SELEX) 2, 3, 4.
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Scheme 1: Basic scheme of SELEX
SELEX begins with a pool of random oligonucleotides flanked with constant sequences at both the 5’ and 3’
ends. The random pool, commonly referred to as a library, is incubated with the desired target. Targets are
extremely varied, and are typically small molecules, proteins, viruses, or cells. The oligonucleotides that do
not bind to the target are washed away, and the binding oligonucleotides are isolated. PCR is used to
amplify the oligonucleotides with desirable binding properties, then the dsDNA PCR product is converted
to ssDNA. That ssDNA makes up the oligonucleotide pool for the subsequent round of selection. The cycle
is repeated approximately eight to twelve times, until a pool of approximately one to one hundred aptamer
sequences are obtained. Sequencing is performed on the newly discovered aptamers, and they can then be
used in applications ranging from pharmaceuticals, biosensing, or biomarker discovery 2, 3, 4.
My honours project consisted of three separate projects, each dealing with a different aspect of aptamer
research. First, I designed and optimized a PCR protocol capable of amplifying ssDNA oligonucleotides
selectively in one step, thereby simplifying the SELEX process. Second, I chemically modified a known and
potentially therapeutic aptamer in an attempt to increase its in vivo stability. Finally, I performed SELEX in
order to discover aptamer pools capable of binding to Vero cells, a well-known immortal mammalian cell
line.
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Design of Linear-After-The-Exponential Polymerase Chain Reaction for SELEX
Background
One of the most challenging steps of SELEX is the isolation of ssDNA from the dsDNA produced during
PCR. Traditionally this step is done using expensive and time-consuming techniques such as exonuclease
degradation or separation via biotinylated primers 5. In the past, Berezovski research group used a two-step
process for amplification and isolation of ssDNA – first traditional symmetric PCR was employed to amplify
dsDNA, then the symmetric PCR product was subjected to asymmetric PCR was used to selectively
amplify ssDNA. While this method was less expensive and time-consuming than most, purification steps
were required and side products were frequently formed.
The goal of this research was to design a novel PCR protocol capable of amplifying ssDNA oligonucleotides
in a single PCR step for use in SELEX. To do this, a technique known as Linear-After-The-Exponential PCR
(LATE-PCR) was employed. LATE-PCR is a special type of asymmetric PCR that allows for efficient
ssDNA production 6, 7, 8. In LATE-PCR, the limiting primer's melting temperature is set higher than the
excess primer's melting temperature. The discrepancy between the limiting primer’s Tm and the excess
primer’s Tm (TmL-Tm
X) allows the limiting primer to bind completely to the DNA template during the
annealing phase. Early in LATE-PCR the amplification curve resembles symmetric PCR showing
exponential growth, and when the limiting primer is depleted the curve becomes linear, resembling
asymmetric PCR 6, 7, 8. This relationship is demonstrated in figure 1, a graph of asymmetric quantitative PCR
(qPCR) data sets in which TmL-Tm
X was variable. In figure 1, the blue curve which represents a TmL-Tm
X of
+5°C is representative of LATE-PCR. It clearly shows a period of exponential growth, followed by a
plateauing linear phase. As well, it shows superior amplification to its counterparts with lower TmL-Tm
X
values, particularly early on in the PCR. Conversely, the yellow, green, and orange curves, which
respectively represent TmL-Tm
X values of +3°C, 0°C, and -3°C do not display as strong an inflection as the
blue curve, and cannot be characterized as LATE-PCR.
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FIGURE 1: QUANTITATIVE PCR DATA MEASURING AMPLIFICATION AS A FUNCTION OF NUMBER OF PCR CYCLES. EACH
CURVE REPRESENTS A SPECIFIC PRIMER SET, EACH WITH A DIFFERENT TML-TM
X. FROM PIERCE ET AL. 2005 6.
It was theorized that the use of LATE-PCR for ssDNA amplification during SELEX could reduce the time
and money required to perform SELEX, contribute to a SELEX protocol that could be automated i.e.
performed by a robotic station. Optimization of LATE-PCR consisted of manipulating variables such as the
ratio of forward primer to reverse primer concentration, total number of PCR cycles, annealing
temperature used during PCR, and type of DNA polymerase using during PCR. The resulting optimized
protocol for a LATE-PCR was not only more efficient and cost-effective than the previous method
employed by Berezovski research group, it tended to produce less undesirable side products and was capable
of amplifying smaller initial concentrations of DNA more effectively than the previously employed method.
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Results & Discussion
Prior to experimentation with LATE-PCR, the ssDNA amplification and isolation method previously
employed by Berezovski research group was assessed. In this method, dubbed “symmetric-then-asymmetric
PCR,” oligonucleotides are first amplified by symmetric PCR to form dsDNA, then the symmetric PCR
product is subjected to asymmetric PCR to selectively amplify ssDNA. We sought to determine the lower
limit of starting DNA concentration it could apparently amplify (limit of detection, LOD), the extent of
amplification, and the extent of side-product formation. The results of this experiment would provide a
basis to judge the effectiveness of future experiments with LATE-PCR.
The results are displayed in figure 2 in the appendix. They show that the LOD of the symmetric-then-
asymmetric method is a starting DNA concentration of 100 pM. PCR reaction mixtures with starting DNA
library concentrations below showed no apparent amplification. At concentrations greater than 100 pM the
symmetric-then-asymmetric method amplified DNA library very effectively, with well over 50% of the
forward primer becoming incorporated into the amplicons. Interestingly, while side product formation was
minimal when the starting DNA concentration was 100 pM, at higher DNA concentrations (1 nM) the
symmetric-then-asymmetric method produced a significant amount of side product. The exact reason for
this is not known, but a possible explanation is that a higher template and amplicon concentration
encourages the formation of complexes – two or more oligonucleotide molecules bound to one another –
which have different electrophoretic mobility than individual oligonucleotides.
Prior to beginning LATE-PCR experiments, a review of literature discussing LATE-PCR was conducted to
determine reasonable experimental conditions to use for the first round of experiments 6, 7, 8. The
theoretical basis behind LATE-PCR is that if the melting temperature of the limiting primer is higher than
that of the excess primer, then during the annealing step of PCR nearly all molecules of the limiting primer
will bind to their template strand, resulting in efficient amplification until the limiting primer has been
exhausted. The general consensus throughout the literature reviewed was that difference in Tm between the
limiting and excess primer (TmL-Tm
X) should be approximately 5-10°C, and that the ideal annealing
temperature used in PCR varies. Using Applied Biosystems® Tm Calculator and knowledge of the typically
used PCR primer sequences for the 40N DNA library, ideal excess (forward) primer and limiting (reverse)
primer sequences for LATE-PCR were designed. The reverse primer typically used for PCR of the 40N
DNA library, a 20 nucleotide sequence with a Tm of 54.03°C, was determined to be appropriate for LATE-
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PCR. The forward primer we decided to use for LATE-PCR is a truncated version of the normal 20
nucleotide primer; it is a 17 nucleotide sequence with a Tm of 48.29°C. The TmL-Tm
X of this primer pair is
5.74°C, which fits the criteria for LATE-PCR.
In addition, the literature reviewed suggested that ratios of excess to limiting primer concentrations can
range from 10 to 100, with 20 to 40 being typical.
The first LATE-PCR experiment consisted of an assay of PCR reactions with varying excess primer to
limiting primer ratios (ratios of 1/20 to 1/80 were analysed) and different annealing temperatures (48-
56°C). The PCR products were analysed with agarose gel electrophoresis, and the gel image is annotated in
figure 3 in the appendix.
The results of this experiment were promising and not unexpected. When the ratio of limiting to excess
primer is small, product formation is low. However, while increasing the ratio of limiting to excess primer
increased product amplification, it also seems to increase the amount of side product produced. Thus a
fairly moderate limiting to excess primer ratio, between 1/40 and 1/20, is likely optimal. As well,
decreasing annealing temperature seemed to slightly increase the yield of both desirable product and side
product. This effect was relatively minor, though annealing temperature required optimization as well.
Another experimental variable emphasized by LATE-PCR literature was the number of PCR cycles used.
The number of cycles used in LATE-PCR is greater than that used in traditional symmetric or asymmetric
PCR, though it is subject to a great deal of variability. The articles reviewed studied the results of LATE-
PCR that ranged from 35 to 85 PCR cycles. Thus our second LATE-PCR experiment consisted of an assay
of PCR reactions with varying numbers of PCR cycles (55-80) and different annealing temperatures (48-
56°C). Due to the concern of side product amplification at very high numbers of PCR cycles, a duplicate
experiment was performed in which none of the PCR mixtures contained any template DNA. This large
negative control experiment serves to determine a “safe” upper limit of PCR cycles in which only template
DNA is amplified. The PCR products were analyzed with agarose gel electrophoresis, and the gel images
are annotated in figures 4 and 5 in the appendix.
The experiments reveal that although higher numbers of cycles yield greater amplification, at 60-65 cycles
unwanted side products begin to be noticeably amplified. Thus 55 was tentatively set as the optimal number
of cycles for LATE-PCR of 40N DNA library.
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The most interesting result from this project came about when the tentatively optimized LATE-PCR
procedure was performed using the truncated 17 nucleotide forward primer, designed especially for LATE-
PCR, and with the normal 20 nucleotide forward primer side-by-side. The only differences in these two
reaction mixtures was the length of the forward primer used and the annealing temperate – the mixture
containing the truncated primer had its annealing time set lower to suit the shorter primer’s decreased Tm.
The mixtures were analysed using agarose gel electrophoresis and the gel image is annotated in the appendix
as figure 6.
It was expected that the PCR mixture containing the specially designed 17 nucleotide LATE-PCR primer
would amplify the template DNA more effectively. However, no noticeable difference existed between the
two PCR mixtures.
The reason that the 20 nucleotide forward primer worked equally as well as the 17 nucleotide forward
primer is still not fully understood. It is possible that at 55 cycles, both of the PCR mixtures’ amplification
reached a linear plateau, and the difference in the extent of amplification is not distinguishable when
quantifying DNA concentration with a method as insensitive as agarose gel electrophoresis. Figure 1 seems
to support this idea. In figure 1, the LATE-qPCR shows superior amplification to the other reactions early
on in PCR, during the LATE-PCR’s exponential phase. However, once the limiting primer of the LATE-
PCR had been exhausted and the slope had become linear, the other reactions’ level of amplification caught
up with that of the LATE-PCR. This idea could be tested with quantitative PCR (qPCR), unfortunately we
did not have the equipment necessary to perform a qPCR experiment.
Following optimization of these essential parameters, a direct comparison of the old symmetric-then-
asymmetric method versus the new LATE-PCR method at varying starting DNA template concentrations
was made. This experiment yielded extremely exciting results. The new LATE-PCR procedure was
superior to the symmetric-then-asymmetric in terms of its amplification yield and LOD, and did not suffer
from an increase in side product formation.
Finally, to ensure that the results of this experiment would be practical and have benefit to PCR, we tested
the newly developed LATE-PCR protocol using a master mix kit from a different company. The reasoning
behind this is that different varieties of DNA polymerase have different strengths. One issues that has been
encountered by members of Berezovski research group is the selection of aptamers and aptamer pools with
high levels of the nucleotides guanine and cytosine, commonly referred to as “high GC”. Although high GC
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oligonucleotides tend to form secondary structures more successfully than oligonucleotides with even
nucleotide distribution, making them likely to be excellent aptamers, their tendency to fold and their
relatively high melting temperature make them difficult to amplify with PCR 9. Some DNA polymerase
varieties are superior at amplifying high GC DNA compared to competitors, and I wanted researchers using
this LATE-PCR protocol to have a back-up option, in case the GoTaq© Polymerase cannot successfully
amplify their aptamer pools due to high GC content. We chose to use the KAPA2G Robust Hotstart PCR
Kit from Kapa Biosystems due to recommendations from other lab members and its relatively low cost. The
results from experimentation with KAPA2G are annotated in figure 8 in the appendix. The KAPA2G DNA
polymerase was shown to be a feasible alternative to GoTaq©’s polymerase, with comparable amplification
and no noticeable side product formation.
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Conclusions
Overall this project was successful. The newly designed LATE-PCR protocol was shown to have superior
efficiency, cost-effectiveness, limit of detection, and level of side product formation compared to the old
symmetric-then-asymmetric PCR method. Following experimentation, formal protocols for LATE-PCR
were written. They can be found in the appendix.
The LATE-PCR protocol was used in Berezovski research group for SELEX experiments for a short time,
though currently we are isolating ssDNA in SELEX experiments by performing exonuclease digestion
following symmetric PCR. The exonuclease method appears to be more robust when it comes to difficult-
to-amplify pools, and is fairly rapid and inexpensive.
In the future, we hope to incorporate LATE-PCR into automated SELEX that will be performed in
Berezovski lab. In automated SELEX, aptamer selection is performed extremely rapidly by a robotic
station. In this circumstance, LATE-PCR will be absolutely ideal for oligonucleotide amplification and
ssDNA isolation because it significantly reduced the number of steps involved in SELEX and streamlines the
process.
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Materials and Methods
Primers and 40N DNA library were obtained from Integrated DNA Technologies (Coralville, USA). The
40N DNA library consists of a mixture of DNA oligonucleotides, each composed of a 40 nucleotide random
sequence flanked by constant 20 nucleotide sequences at each end that serve as primer regions. Both the full
forward primer (5’-CTC CTC TGA CTG TAA CCA CG-3’) and truncated forward primer (5’- CTC CTC
TGA CTG TAA CC-3’) were labelled with FAM on their 5’ ends, whereas the reverse primer (5’-GGC-
TTC-TGG-ACT-ACC-TAT-GC-3’) was unlabelled.
Primer annealing temperatures were calculated with Applied Biosystems® Tm Calculator
(www6.appliedbiosystems.com/support/techtools/calc) from Life Technologies (Carlsbad, USA).
Several varieties of DNA polymerases and master mixes were used throughout this project. The most
frequently used was the GoTaq© Hot Start PCR Kit from Promega (Madison, USA). The master mixes
prepared from this kit contained nuclease-free ddH2O, 1X GoTaq© Flexibuffer, 2.5 mM MgCl2, 250 μM
dNTPs, 0.025 UμL-1 GoTaq© Hot Start DNA Polymerase, and varying amounts of forward and reverse
primer. In addition to the Promega master mixes and DNA polymerase, the KAPA2G Robust Hotstart PCR
Kit from Kapa Biosystems (Wilmingtom, USA) was used. Manufacturers’ instructions were followed when
preparing the KAPA2G master mixes. All PCR reactions were carried out using the vapo.protect
Mastercycler proS from Eppendorf (Hamburg, Germany).
Gel electrophoresis experiments were performed in 0.5X TAE buffer (20 mM Tris, 10 mM acetic acid, and
0.5 mM EDTA), which had been prepared in-lab. UltraPureTM Agarose was obtained from Invitrogen
(Carlsbad, USA). Gels were visualized using the Fluorochem® Q from Alpha Innotech (Santa Clara, USA).
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Assessment of Current Method – Symmetric-then-Asymmetric PCR
Two master mixes were prepared – one for symmetric PCR, one for asymmetric PCR. Their components
are as follows:
Symmetric master mix:
1X Colourless GoTaq® Flexi Buffer (Promega), from 5X concentration stock
2.5 mM MgCl2 (Promega)
300 nM reverse DNA primer (sequence: 5'-GGC TTC TGG ACT ACC TAT GC-3')
300 nM 5' FAM-labelled forward primer (sequence: 5'-CTC CTC TGA CTG TAA CCA CG-3')
200 uM dNTPs
0.025 UuL-1 Gotaq® Hot Start DNA Polymerase (Promega)
Asymmetric master mix:
1X Green GoTaq® Flexi Buffer (Promega), from 5X concentration stock
2.5 mM MgCl2 (Promega)
50 nM reverse DNA primer (sequence: 5'-GGC TTC TGG ACT ACC TAT GC-3')
1 µM 5' FAM-labelled forward primer (sequence: 5'-CTC CTC TGA CTG TAA CCA CG-3')
200 uM dNTPs
0.025 UuL-1 Gotaq® Hot Start DNA Polymerase (Promega)
Eight 45 µL aliquots of the symmetric master mix were placed into 0.2 mL PCR tubes. 5 µL of DNA
library solution was placed into each of the eight master mix aliquots. The DNA concentration for each
DNA library solution used varied (1 aM, 10 aM, 1 fM, 100 fM, 1 pM, 100 pM, 1 nM, and 0 (negative
control) were used).
The following PCR program was used for both symmetric and asymmetric amplification:
Initial denaturation: 2 minutes at 94º C
Followed by 15 cycles of the following steps:
Denaturation: 30 seconds at 94º C
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Annealing: 15 seconds at 56º C
Extension: 15 seconds at 72º C
Hold at 4º C
Eight 45 µL aliquots of the asymmetric master mix were placed into 0.2 mL PCR tubes. 5 µL of each
symmetric PCR product was placed into one of the eight asymmetric master mix aliquots. Asymmetric
amplification was completed using the same PCR program as was used for symmetric amplification.
A 3% agarose gel in 0.5X TAE buffer was prepared, and 10 µL of each symmetric-then-asymmetric PCR
products was loaded into the gel. The gel was then run at 150 V for 30 minutes. The gel image is annotated
in figure 2 in the appendix.
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Initial Optimization of Primer Ratios
An assay was performed in which the limiting (reverse) primer concentration and the annealing
temperature were simultaneously varied. Three master mixes were prepared, described below:
1X Green GoTaq® Flexi Buffer (Promega), from 5X concentration stock
2.5 mM MgCl2 (Promega)
12.5 nM, 25 nM, or 50 nM reverse DNA primer (sequence: 5'-GGC TTC TGG ACT ACC TAT
GC-3')
1 µM 5' FAM-labelled forward primer (sequence: 5'-CTC CTC TGA CTG TAA CCA CG-3')
400 uM dNTPs
0.025 UuL-1 Gotaq® Hot Start DNA Polymerase (Promega)
Five 12.5 µL aliquots of each master mix were placed into 0.2 mL PCR tubes. 2.5 µL 100 nM DNA library
was placed into four out of five aliquots of each master mix, and to the remaining DNA library-free master
mix aliquots 5 µL ddH2O was added.
Using the gradient function of the vapo.protect Mastercycler proS, the PCR program was set such that the
annealing temperature varied. The PCR program is described below:
Initial denaturation: 2 minutes at 95º C
Followed by 55 cycles of the following steps:
Denaturation: 10 seconds at 94º C
Annealing: 20 seconds at 48, 50, 52, 54 or 56º C
Extension: 10 seconds at 72º C
Hold at 4º C
Note that for the negative controls the annealing temperature was set at 52°C.
A total of 18 samples were prepared, each with a different combination of primer ratio and annealing
temperature.
A 3% agarose gel in 0.5X TAE buffer was prepared, and 10 µL of each PCR products was loaded into the
gel. The gel was then run at 150 V for 45 minutes. The gel image is annotated in figure 3 in the appendix
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Initial Optimization of Number of PCR Cycles
An assay was performed in which the number of PCR cycles and the annealing temperature were
simultaneously varied. One master mix was prepared, described below:
1X Green GoTaq® Flexi Buffer (Promega), from 5X concentration stock
2.5 mM MgCl2 (Promega)
25 nM reverse DNA primer (sequence: 5'-GGC TTC TGG ACT ACC TAT GC-3')
1 µM 5' FAM-labelled forward primer (sequence: 5'-CTC CTC TGA CTG TAA CC-3')
400 uM dNTPs
0.025 UuL-1 Gotaq® Hot Start DNA Polymerase (Promega)
A total of 48 samples were prepared. 48 12.5 µL aliquots of master mix were prepared. 2.5 µL 100 nM
DNA library was added to each of 24 master mix aliquots. 2.5 µL ddH2O was added to the remaining
aliquots.
Using the gradient function of the vapo.protect Mastercycler proS, the PCR program was set such that the
annealing temperature varied. The PCR program is described below:
Initial denaturation: 2 minutes at 95º C
Followed by 55 to 80 cycles of the following steps:
Denaturation: 10 seconds at 94º C
Annealing: 20 seconds at 48, 50, 52, or 54 º C
Extension: 10 seconds at 72º C
Hold at 4º C
Two 3% agarose gels in 0.5X TAE buffer were prepared, and 10 µL of each PCR products was loaded into
a gel. The gels were then run at 150 V for 55 minutes. The gel images are annotated in figures 4 and 5 in
the appendix.
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Selection of a Primer Pair
Two master mixes were prepared. The only difference between them was the type of forward primer each
contained. One contained the regular 20 nucleotide primer, the other contained the truncated 17
nucleotide forward primer designed for LATE-PCR. The master mix components are listed below.
1X Green GoTaq® Flexi Buffer (Promega), from 5X concentration stock
2.5 mM MgCl2 (Promega)
25 nM reverse DNA primer (sequence: 5'-GGC TTC TGG ACT ACC TAT GC-3')
1 µM 5' FAM-labelled forward primer (sequence: either 5'-CTC CTC TGA CTG TAA CC-3' or 5'-CTC
CTC TGA CTG TAA CCA CG-3')
400 uM dNTPs
0.025 UuL-1 Gotaq® Hot Start DNA Polymerase (Promega)
Two 12.5 µL aliquots of each master mix were prepared. 2.5 µL 100 nM DNA to one aliquot of each
master mix variety. 2.5 µL ddH2O was added to each of the remaining two master mix aliquots.
Using the gradient function of the vapo.protect Mastercycler proS, the PCR program was set such that the
annealing temperature varied. The PCR program is described below:
Initial denaturation: 2 minutes at 95º C
Followed by 55 cycles of the following steps:
Denaturation: 10 seconds at 94º C
Annealing: 20 seconds at 52 or 56 º C (note: the samples containing the 20 nucleotide
primer had their annealing temperature set to 56° C, the samples with the 17 nucleotide
primer had an annealing temperature of 52° C.)
Extension: 10 seconds at 72º C
Hold at 4º C
A 3% agarose gels in 0.5X TAE buffer was prepared, and 10 µL of each PCR products was loaded into the
gel. The gel was then run at 150 V for 25 minutes. The gel image is annotated in figure 6 in the appendix.
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Direct Comparison of LATE-PCR and Symmetric-then-Asymmetric PCR
In this experiment, both LATE-PCR and symmetric-then asymmetric PCR were performed, and their
results were assessed side-by-side using agarose gel electrophoresis.
For the LATE-PCR, the first step was to make a master mix, whose components are listed below:
1X Green GoTaq® Flexi Buffer (Promega), from 5X concentration stock
2.5 mM MgCl2 (Promega)
25 nM reverse DNA primer (sequence: 5'-GGC TTC TGG ACT ACC TAT GC-3')
1 µM 5' FAM-labelled forward primer (sequence: 5'-CTC CTC TGA CTG TAA CCA CG-3')
400 uM dNTPs
0.025 UuL-1 Gotaq® Hot Start DNA Polymerase (Promega)
Seven 25 µL aliquots of this master mix were prepared. Each aliquot was combined with 5 µL of a DNA
library solution. The DNA concentration for each DNA library solution used varied (10 fM, 100 fM, 1 pM,
10 pM, 100 pM, 1 nM, and 0 (negative control) were used).
The seven PCR mixtures were amplified according to the following PCR program:
Initial denaturation: 2 minutes at 95º C
Followed by 55 cycles of the following steps:
Denaturation: 10 seconds at 94º C
Annealing: 20 seconds at 56 º C
Extension: 10 seconds at 72º C
Hold at 4º C
The LATE-PCR mixtures were then set aside briefly while the symmetric-then-asymmetric PCR mixtures
were prepared.
For the symmetric-then asymmetric-PCR, two master mixes were prepared – one for symmetric PCR, one
for asymmetric PCR. Their components are as follows:
Symmetric master mix:
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1X Colourless GoTaq® Flexi Buffer (Promega), from 5X concentration stock
2.5 mM MgCl2 (Promega)
300 nM reverse DNA primer (sequence: 5'-GGC TTC TGG ACT ACC TAT GC-3')
300 nM 5' FAM-labelled forward primer (sequence: 5'-CTC CTC TGA CTG TAA CCA CG-3')
200 uM dNTPs
0.025 UuL-1 Gotaq® Hot Start DNA Polymerase (Promega)
Asymmetric master mix:
1X Green GoTaq® Flexi Buffer (Promega), from 5X concentration stock
2.5 mM MgCl2 (Promega)
50 nM reverse DNA primer (sequence: 5'-GGC TTC TGG ACT ACC TAT GC-3')
1 µM 5' FAM-labelled forward primer (sequence: 5'-CTC CTC TGA CTG TAA CCA CG-3')
200 uM dNTPs
0.025 UuL-1 Gotaq® Hot Start DNA Polymerase (Promega)
Seven 25 µL aliquots of the symmetric master mix were placed into 0.2 mL PCR tubes. 5 µL of DNA
library solution was placed into each of the seven master mix aliquots. The DNA concentration for each
DNA library solution used varied (10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM, and 0 (negative control)
were used).
The following PCR program was used for both symmetric and asymmetric amplification:
Initial denaturation: 2 minutes at 94º C
Followed by 15 cycles of the following steps:
Denaturation: 30 seconds at 94º C
Annealing: 15 seconds at 56º C
Extension: 15 seconds at 72º C
Hold at 4º C
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Eight 45 µL aliquots of the asymmetric master mix were placed into 0.2 mL PCR tubes. 5 µL of each
symmetric PCR product was placed into one of the eight asymmetric master mix aliquots. Asymmetric
amplification was completed using the same PCR program as was used for symmetric amplification.
A 3% agarose gel in 0.5X TAE buffer was prepared, and 10 µL of each of the 14 samples was loaded into
the gel. The gel was then run at 150 V for 50 minutes. The gel image is annotated in figure 7 in the
appendix.
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Verification of LATE-PCR Feasibility with a Different Variety of DNA Polymerase
A master mix was prepared with the following components:
1X KAPA2G Buffer A, from 5X concentration stock
25 nM reverse DNA primer (sequence: 5'-GGC TTC TGG ACT ACC TAT GC-3')
1 uM 5' FAM-labelled forward primer (sequence: 5'-CTC CTC TGA CTG TAA CCA CG-3')
400 uM dNTPs
0.025 UuL-1 KAPA2G Robust Hot Start DNA Polymerase
Two 25 µL aliquots of the master mix was prepared. To one aliquot, 5 µL 100 nM DNA library was added.
To the other, 5 µL ddH2O was added.
The two PCR mixtures were subjected to the following PCR program:
Initial denaturation: 30 seconds at 95º C
Followed by 40 cycles of the following steps:
Denaturation: 10 seconds at 95º C
Annealing: 20 seconds at 56º C
Extension: 10 seconds at 72º C
Hold at 4º C
A 3% agarose gel in 0.5X TAE buffer was prepared, and 10 µL of each of the two samples was loaded into
the gel. The gel was then run at 150 V for 30 minutes. The gel image is annotated in figure 8 in the
appendix.
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Improving In Vivo Stability of Oncolytic Virus-Protecting Aptamers through PEGylation
Background
The first effective cancer-treating drugs were discovered in the early 20th century, and millions of lives have
been saved by chemotherapy since that time. Early chemotherapies such as nitrogen mustard and
methotrexate, derived from the warfare agent mustard gas, were notoriously toxic, brutal drugs to be
treated with 10. While modern chemotherapies are not as toxic as many of their predecessors, they still carry
side effects lists that include extreme nausea, immune suppression, and secondary cancers. The extreme
side effects of chemotherapies derive from the fact that they are designed to kill cancers cells, but do so with
imperfect selectivity, thus they cause a high level of damage to healthy cells as well.
Modern research into new cancer treatments is largely focused on improving selectivity of drugs to cancer
cells, and one novel and extremely promising approach is the use of oncolytic viruses (OVs). OVs are a
diverse group of wild-type and genetically engineered viruses that are capable of selectively destroying
cancer cells in a variety of ways 11, 12. One such virus is enhanced vesicular stomatitis virus (VSVΔ51).
VSVΔ51 belongs to the Rhabdoviridae family of viruses, and contains a mutation causing deletion of a single
amino acid in its viral matrix protein which confers its ability to selectively kill cancerous cells. While
VSVΔ51 is capable of infecting mammals, because it is extremely sensitive to interferons, a class of proteins
produced in response to pathogens, infections are typically mild. However, because cancer cells often have
defective interferon pathways, VSVΔ51 is able to cause lethal infections in a wide range of cancer cells 13, 14.
The primary challenge of working with OVs as cancer therapeutics in poor in vivo half-life due to
destruction by neutralizing antibodies (nAbs) of the immune system. To overcome this problem, Berezovski
research group has investigated Aptamer-Facilitated VIrus Protection (AptaVIP), which involves the use of
aptamers capable of binding to VSVΔ51’s outer surface and shielding virus particles from neutralization by
nAbs. Several aptamer sequences capable of shielding VSVΔ51 from nAbs have been elucidated by
Berezovski research group14. Past cell-based viral infectivity assays performed by Berezovski research group
have shown that these aptamer sequences are capable of shielding VSVΔ51 from nAbs in vitro, enabling
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VSVΔ51 to infect and kill interferon-deficient cells 14. However, aptamers are subject to degradation by
nucleases when used in vivo. This degradation is rapid, and reduces the half-life of aptamers to only a few
hours, making unmodified aptamers unfeasible for in vivo applications 16, 17. Fortunately, many methods of
modifying aptamers exist to confer in vivo stability to aptamers, allowing them to be used therapeutically 16,
17.
This project involved the covalent attachment of a polyethylene glycol (PEG) moiety to the VSVΔ51-
binding aptamer ZMYK-23. The covalent attachment of a PEG group to another molecule is commonly
referred to as PEGylation. PEGylation is a common modification in pharmaceutical science, being used to
improve delivery and stability of small molecule, peptide, protein, and oligonucleotide drugs 17, 18.
Numerous PEGylated drugs are currently on the market, notable among them are Pegasys® (Hoffman-La
Roche, Inc., USA), an interferon for hepatitis treatment, Neulasta® (Amgen, USA), a protein used to
stimulate neutrophil growth in patients undergoing chemotherapy, and Macugen® (OSI Pharmaceuticals,
USA), an aptamer for treatment of macular degeneration 17, 18, 19.
This project involved several phases with the ultimate goal of producing PEGylated aptamers capably of
effective VSVΔ51 AptaVIP in vivo. First, a protocol for PEGylating aptamers efficiently was designed and
optimized. Following that, the PEGylated aptamers needed to be isolated and purified. Then cell-based
viral infectivity assays were performed to determine whether or not PEGylated aptamers could successfully
perform AptaVIP in vitro. In addition, flow cytometry was employed to study the aptamers’ binding
characteristics. Finally, had in vitro experiments been successful, we would have performed in vivo
experiments to show if VSVΔ51 AptaVIP with PEGylated aptamers has therapeutic potential.
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Results & Discussion
The first task required for this project was to design an organic reaction capable of efficiently linking the
VSVΔ51-binding aptamer ZMYK-23 to PEG. Factors including the type of linkage, reactant concentrations,
solvent systems, pH, and reaction time and temperature had to be optimized. Several publications7, 8, 9, 10
were reviewed in order to determine appropriate starting conditions. Based on articles describing similar
PEG conjugation reactions and PEGylation in pharmacology, the reactants selected were 20 kDa mPEG
modified with succinimidyl carbonate (mPEG-SC 20K) and 3’ amino modified aptamer. The conjugation
mechanism shown in scheme 2.
In the early part of this task, results were modest. Some factors, such as buffer, reaction mixture pH, and
acetonitrile concentration seem to have a small effect on the extent of aptamer PEGylation (see figures 10,
11, 12, and 14 in the appendix). At the same time, many factors that we predicted could impact
PEGylation, such as reaction time, temperature, and agitation seemingly had no effect on PEG-aptamer
yield (see figures 9 and 13 in the appendix). Eventually a plateau seemed to have been reached, as
PEGylation yield never rose above ~25%, despite experimentation with several different variables. While
these results were frustrating while attempting to design a reaction with a high PEGylation yield, they were
quite informative regarding the nature of the reaction. The lack of impact of reaction temperature,
agitation, and time indicated that this reaction is kinetically very favourable, going to completion rapidly
and without the need for added internal energy to reach activation barriers. In addition, one might expect
that reagent degradation might contribute to the seemingly identical PEGylation yields of both 4 hour and
20 hour incubated reaction mixtures. Indeed, according to the manufacturers, the mPEG-SC used in this
experiments has a hydrolysis half-life of 20.4 minutes at pH 8, 25°C.
The breakthrough came when an assay of reaction mixtures with varying reagent concentrations was
assessed. Our 25% yield plateau was shown to be only a temporary phase, as increasing the concentration of
amino-modified ZMYK-23 to 4 to 10 µM pushed the PEGylation yield up to ~75% (see figure 15B and
15C). Interestingly, the correlation between reagent concentration and yield in this reaction is not
consistent, as figure 16 reveals that at increasing aptamer concentration above ~30 µM causes a drastic
reduction in PEGylation yield. The reason for this effect is not known, but some speculations can be made.
Le Chatalier’s Principle cannot explain the increased PEGylation yield at high reagent concentrations, as
both the aptamers and PEG concentrations were increased. It is possible that the PEGylation reaction is
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completing with mPEG-SC degradation, and by increasing reagent concentration, the number of collisions
between the aptamer’s amino group and the succinimidyl carbonate group on the PEG molecule is
increased and PEGylation yield in turn increases. The lack of reactivity at high reagent concentrations is
likely due to the effect of PEG on solution viscosity and reagent mobility, and possibly also due to inter-
aptamer complexation, leading to steric hindrance preventing the reaction between the aptamer’s amino
group and the PEG’s succinimidyl carbonate group.
Once a reasonable PEGylation yield could be achieved, the next step was to isolate and purify the
PEGylated aptamer. Many effective methods exist for the purification of unmodified nucleic acids.
However, the bulky PEG moiety on PEG-ZMYK-23 alters its physical and chemical properties, making
many conventional nucleic acid purification methods non-ideal, and making the task of PEG-ZMYK-23
isolation quite challenging. PEG-modified pharmaceuticals are typically isolated using preparative High
Performance Liquid Chromatography (prep HPLC) 17. However, prep HPLC facilities are expensive and
difficult to find. I hoped to devise a way of isolating an adequate amount of PEGylated aptamer for in vitro
experiments, and possibly look into prep HPLC if in vitro experiments were successful and in vivo
experiments were a logical next step.
The purification technique I found was electroelution, a fairly uncommon technique in which a slice of gel
from gel electrophoresis containing the desired biomolecule is subjected to an electric current, causing the
biomolecule in question to migrate out of the gel 21. Unlike most DNA purification techniques, which rely
on the solubility characteristics of DNA, electroelution relies only on charge to isolate DNA. This makes it
an ideal method for isolating nucleic acids that have been modified with bulky moieties such as 20 kDa PEG.
Figure 17 in the appendix shows the result of electroelution of PEGylated ZMYK-23. 50 µL 200 nM
PEGylated ZMYK-23 was successfully produced. While this was a good result, the total amount of
PEGylated aptamer collected was modest, and would not be sufficient for in vivo experimentation.
Therefore, we kept prep HPLC in mind as a possibility in the future.
Once we had developed a method for producing and isolating PEGylated aptamer, we moved on to cell-
based experimentation. The first step was to titre polyclonal anti-VSV∆51 nAbs, to determine an
appropriate dilution to use in cell-based viral infectivity assays. Unfortunately, the stock of anti-VSV∆51
nAbs previously used by Berezovski research group in VSV∆51 AptaVIP experiments, which had been
produced by bunny #2, has denatured and was no longer biologically active. We received a new stock of
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anti-VSV∆51 nAbs, produced by bunny #3, from Dr. John Bell and did all subsequent experimentation
with that stock. Results of the titring experiment are displayed in figure 18 in the appendix. They show that
a dilution of 1/5000 is the smallest concentration of nAbs capable of eliminating all infectivity from
VSV∆51 under the experimental conditions employed, therefore 1/5000 the ideal dilution of nAbs to use
in future experimentation.
Next, cell-based viral infectivity assays were performed to verify that unmodified aptamers were capable of
shielding VSV∆51 from nAbs. The results of this experiment can be found in figure 19 in the appendix.
This experiment was repeated many times under many different experimental conditions, but at no point
did the VSV-binding aptamers previously selected by Berezovski research group display any capacity to
shield VSV∆51 from nAbs. This result was disappointing and quite surprising. The aptamers in question had
previously been shown to perform VSV∆51 AptaVIP effectively in cell-based viral infectivity assays 14,
therefore it was assumed that they would continue to work throughout this project.
To attempt to explain this unexpected outcome, we considered the variables present in the experiment,
and what had changed between my cell-based viral infectivity assays and equivalent ones previously
performed in Berezovski lab. The most significant variable seemed to be the nAbs. The aptamers used in the
experiment had been selected using anti-VSV∆51 nAbs isolated from bunny #2 in Dr. John Bell’s lab.
However, I had performed cell-based viral infectivities using nAbs from a different animal, bunny #3. We
hypothesized that nAbs from the different animals may bind to different epitopes on the virus. If this was
the case, it is possible that while the aptamers being used could shield VSV∆51 from nAbs produced by
bunny #2, nAbs from other animals would not be hindered by those aptamers.
To test this idea, we performed a flow cytometric displacement assay. In this assay, VSV∆51 particles were
first incubated with a fluorescently labelled polyclonal mixture of VSV∆51-binding aptamers, and the
extent of binding was measured with flow cytometry. Then nAbs from bunny #3 were added to the
VSV∆51 and aptamer-containing sample, and flow cytometry was used to determine to what extent the
nAbs could displace the aptamers. A high level of displacement would indicate that the aptamers and the
nAbs from bunny #3 bound to the same epitopes on VSV∆51. However, if the VSV particles were still
bound to a significant amount of fluorescent aptamer after being incubated with nAbs, it is likely that the
nAbs bind to different epitopes than the aptamers. The flow cytometric histogram is displayed in figure 20
in the appendix.
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The extent of aptamer binding to the virus decreases after exposure to nAbs from bunny #3, but there is
still a significant amount of fluorescent aptamer still attached to VSV∆51 after incubation with nAbs.
Though this result is not incontrovertible, it is suggestive that the polyclonal aptamer mixture employed
binds to several different epitopes on the surface of VSV∆51 than do the anti-VSV∆51 nAbs isolated from
bunny #3. Some factors that might have contributed to the decreased level of fluorescence detected on the
VSV∆51 after incubation with nAbs include loss of aptamer from the mixture from the extra wash
following nAbs incubation, as well as some overlap in epitopes between the polyclonal aptamer mixture and
the polyclonal nAbs.
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Conclusions
We were unable to demonstrate the feasibility of using PEGylated VSV∆51-binding aptamers in AptaVIP,
thus we failed to achieve the goal of this research project. We did, however, glean a good deal of interesting
information and hopefully contribute to future research prospectives. The development of a method for
producing and isolating PEGylated aptamers will likely be valuable to future research projects. PEGylation
is a fairly common process in aptamer research, therefore it is not unreasonable to expect that in the future
a researcher with connections to Berezovski research group may be able to use our PEGylation
experimental results to successfully inform their own PEGylation experimental design.
The cell-based viral infectivity assay and flow cytometric displacement assay also yielded fascinating results
worth investigating further in the future. They demonstrated that the VSV∆51-binding and protecting
aptamers are not necessarily able to protect against nAbs they had not been selected to protect against. This
results encourages us to explore the binding that occurs between nAbs, VSV∆51-binding aptamers, and
epitopes on the surface of VSV∆51. The flow cytometric results suggest that there are differences between
the sites that known VSV∆51-binding aptamers bind and the epitopes that polyclonal nAbs from bunny #2
bind, however we have not determined if there are any overlapping sites. In the future, flow cytometric
displacement assays that analyze the displacement of individual monoclonal VSV∆51-binding antibodies by
anti-VSV∆51 nAbs produced by a variety of different animals could potentially yield extremely fascinating
and valuable information. Such experiments could determine which aptamers are the most effective at
protecting VSV∆51 from nAbs from a wide range of animals, which could eventually lead to development
of highly effective, versatile aptamer pools for VSV∆51 AptaVIP that could potentially be used clinically. It
also opens up the possibility of research directly into the epitopes that anti-VSV∆51 nAbs bind to. Such
research is within the field of proteomics and is quite ambitious, but discovery of epitopes important to
VSV∆51 neutralization makes possible novel research into designing smarter, more targeted methods for
extending VSV∆51’s in vivo half-life and increasing VSV∆51’s therapeutic potential.
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Materials & Methods
Polyethylene glycol (mPEG-succinimidyl carbonate 20 kDa) was obtained for Laysan Bio Inc. (Arab, USA).
All aptamers were obtained from Integrated DNA Technologies (Coralville, USA). Aptamer sequences are
displayed in table 1 below.
Table 1: List of all VSV-binding aptamer sequences used in this research. All sequences were discovered by members of
Berezovski research group.
Aptamer name Aptamer sequence (5’ – 3’)
ZMYK-21 CTC CTC TGA CTG TAA CCA CGC GGG AAC CAA ATC ACG TCC TAG
ATT GTG ATG AAC CTC GGC GCA TAG GTA GTC CAG AAG CC
ZMYK-22 CTC CTC TGA CTG TAA CCA CGG CGA CAA CAC GGA CGG TTG AGA
CTT TAA TTC TGC TCA CGG GCA TAG GTA GTC CAG AAG CC
ZMYK-23 CTC CTC TGA CTG TAA CCA CGG GGA CCT ATC AGG CGA TGT GAA
AAC TCT TAT ACC ACT GGG CAT AGG TAG TCC AGA AGC C
ZMYK-29 CTC CTC TCT GTA ACC ACG CAC ATC CTA CGT TTG CCA CGC GCT
ACT CCG CCA TCT ACC CGC ATA GGT AGT CCA GAA G
MS-50 CTC CTC TGA CTG TAA CCA CGC CAT CAC CCT ATT ATC TCA TTA
TCT CGT TTT CCC TAT GCG GCA TAG GTA GTC CAG AAG CC
SS-31 CTC CTC TGA CTG TAA CCA CGT GAC CCG AGA TTC TAG TGA TTG
CTT GTT CGG TAT GTT CGG CAT AGG TAG TCC AGA AGC C
SS-37 CTC CTC TGA CTG TAA CCA CGG CAT AGC GGG GGA GAT GGG GGA
TGA CTT GGG TGT GAT GGG GCA TAG GTA GTC CAG AAG CC
SS-39 CTC TCC TCT GAC TGT AAC CAC GGC ACT TCA CTT CTC CTC TGA
CTG TAA CCA CGC GCA TAG GTA GTC CAG AAG CA
Note that aptamers that were ordered with the intention of being conjugated with PEG were modified with
an amino group on their 3’ terminal.
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Gel electrophoresis experiments were performed in 0.5X TAE buffer (20 mM Tris, 10 mM acetic acid, and
0.5 mM EDTA), which had been prepared in-lab. UltraPureTM Agarose was obtained from Invitrogen
(Carlsbad, USA). Gels were visualized using the Fluorochem® Q from Alpha Innotech (Santa Clara, USA).
Several products were purchased for attempted PEGylated aptamer purification. VWR® Centrifugal Filters
(modified PES, 30K, 500 µL) were obtained from VWR International (Radnor, USA). ZebaTM Spin
Desalting Columns (40K MWCO, 2 mL) were obtained from Thermo Scientific (Waltham, USA). D-TubeTM
Dialyser Midi MWCO 6-8 kDa tubes were used for electroelution and were obtained from EMD Millipore
(Billerica, USA).
Original samples of Vero cells, VSV∆51-YFP, and polyclonal anti-VSV neutralizing antibodies from bunny
#2 were obtained from Dr. John C. Bell’s laboratory at the Ottawa Hospital Research Institute (Ottawa,
Canada). Samples of VSV∆51-YFP (conc. 1×1010 pfu/mL) were prepared by myself and Darija
Muharemagic by infecting Vero cells with VSV∆51-YFP and purifying the virus through sucrose cushion
purification, as described in Diallo et al., 2012 22.
The medium used for all cell cultures was Dulbecco’s Modified Eagle Medium (DMEM) + 10% FBS
(HyClone, Thermo Scientific, Waltham, USA), and the cultures were grown in HeracellTM 150i CO2
incubator (Thermo Scientific, Waltham, USA). Vero cell cultures were grown and maintained according to
Ammerman et al., 2008 23 with the exception that the cultures were grown in 150 mm dishes from BD
Biosciences (Franklin Lakes, USA) rather than 75 cm2 cell cultures flasks.
Flow cytometric analysis was performed using the Beckman Coulter Cytomics FC500 and flow cytometric
data was analysed using Kaluza® Flow Analysis Software from Beckman Coulter (Mississauga, Canada). Yeast
ribonucleic acid was obtained from Calbiochem® (San Diego, USA). Dulbecco’s phosphate buffered saline
(DPBS) with low Mg2+ and low Ca2+ (HyClone, Thermo Scientific, Waltham, USA).
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Optimization of Conjugation of PEG to ZMYK-23
First Trial – Determining the Effect of Reaction Time on Aptamer PEGylation
The reaction mixture was a solution of 2 μM ZMYK-23 (5’ FAM, 3’ amino) and 200 μM mPEG-SC 20K in
DPBS. One reaction mixture was prepared and incubated at 30°C and 400 rpm for 4 hours, and a second
reaction mixture was prepared and incubated at 30°C and 400 rpm for 20 hours. The PEGylation results
were assessed through agarose gel electrophoresis. The gel was 1.5% agarose in 0.5X TAE buffer and was
run at 150 V for 30 minutes. The gel image is annotated in figure 9 in the appendix.
Scheme 2: Mechanism of conjugation between primary amine and succinimidyl carbonate
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Determining the Effect of Reaction Mixture Buffer and pH on Aptamer PEGylation – Acetonitrile and
Sodium Carbonate
The reaction mixture was a solution of 2 μM ZMYK-23 (5’ FAM, 3’ amino) and 200 μM mPEG-SC 20K in
a solvent system of one half acetonitrile (commonly written as MeCN), one half sodium carbonate buffer
(100 mM Na2CO3, pH 11). It was incubated at ambient temperature for one hour, then characterized using
agarose gel electrophoresis. The gel was 1.5% agarose in 0.5X TAE buffer and was run at 150 V for 25
minutes. The gel image is annotated in figure 10 in the appendix.
Scheme 2: Mechanism of conjugation between primary amine and succinimidyl carbonate
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Determining the Effect of Reaction Mixture Buffer and pH on Aptamer PEGylation – Acetonitrile and
Borate Buffered Saline
The reaction mixture was a solution of 1 μM ZMYK-23 (5’ FAM, 3’ amino) and 100 μM mPEG-SC 20K in
a solvent system of one half acetonitrile, one half borate buffered saline (BBS) solution (10 mM Na2B4O7,
150 mM NaCl, pH 9). It was incubated at ambient temperature for one hour, then characterized using
agarose gel electrophoresis. The gel was 1.5% agarose in 0.5X TAE buffer and was run at 200 V for 25
minutes. The gel image is annotated in figure 11 in the appendix.
Scheme 2: Mechanism of conjugation between primary amine and succinimidyl carbonate
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Determining the Effect of Acetonitrile Concentration on Aptamer PEGylation
An assay of PEGylation reaction mixtures with varying concentrations of acetonitrile was assembled. The
reaction mixtures were solutions of 1 μM ZMYK-23 (5’ FAM, 3’ amino) and 100 μM mPEG-SC 20K in a
solvent systems of acetonitrile and BBS (10 mM Na2B4O7, 150 mM NaCl, pH 9). Three different
acetonitrile to BBS ratios were tested – 1:3, 1:1, and 3:1. The reaction mixtures were incubated at ambient
temperature for one hour, then characterized using agarose gel electrophoresis. The gel was 1.5% agarose
in 0.5X TAE buffer and was run at 200 V for 25 minutes. The gel image is annotated in figure 12 in the
appendix.
Scheme 2: Mechanism of conjugation between primary amine and succinimidyl carbonate
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Determining the Effect of Heat and Agitation on Aptamer PEGylation
The reaction mixture was a solution of 1 μM ZMYK-23 (5’ FAM, 3’ amino) and 100 μM mPEG-SC 20K in
a solvent system of one half acetonitrile, one half borate buffered saline (BBS) solution (10 mM Na2B4O7,
150 mM NaCl, pH 9). It was incubated 50°C, 300 rpm in an Eppendorf Thermomixer for one hour, then
characterized using agarose gel electrophoresis. The gel was 1.5% agarose in 0.5X TAE buffer and was run
at 200 V for 20 minutes. The gel image is annotated in figure 13 in the appendix.
Scheme 2: Mechanism of conjugation between primary amine and succinimidyl carbonate
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Determining the Effect of Different Organic Solvents of Aptamer PEGylation
Two reaction mixtures were prepared. The first was a solution of 1 μM ZMYK-23 (5’ FAM, 3’ amino) and
100 μM mPEG-SC 20K in a solvent system of one half DMSO, one half borate buffered saline (BBS)
solution (10 mM Na2B4O7, 150 mM NaCl, pH 9). The second was a solution of 1 μM ZMYK-23 (5’ FAM,
3’ amino) and 100 μM mPEG-SC 20K in a solvent system of one half acetone, one half borate buffered
saline (BBS) solution (10 mM Na2B4O7, 150 mM NaCl, pH 9). They were incubated at ambient
temperature for one hour, then characterized using agarose gel electrophoresis. The gel was 1.5% agarose
in 0.5X TAE buffer and was run at 200 V for 30 minutes. The gel image is annotated in figure 14 in the
appendix.
Scheme 2: Mechanism of conjugation between primary amine and succinimidyl carbonate
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Determining the Effect of Reagent Concentration on Aptamer PEGylation
Three reaction mixtures were prepared. The first was a solution of 2 μM ZMYK-23 (5’ FAM, 3’ amino)
and 200 μM mPEG-SC 20K. The second was a solution of 4 μM ZMYK-23 (5’ FAM, 3’ amino) and 400
μM mPEG-SC 20K. The third was a solution of 10 μM ZMYK-23 (5’ FAM, 3’ amino) and 1 mM mPEG-
SC 20K. The solvent system for all three mixtures was one half acetonitrile, one half borate buffered saline
(BBS) solution (10 mM Na2B4O7, 150 mM NaCl, pH 9). They were each incubated for one hour at ambient
temperature, then characterized using agarose gel electrophoresis. The gels were 1.5% agarose in 0.5X
TAE buffer and were each run at 200 V for 20 minutes. The gel images are annotated in figure 15 in the
appendix.
Scheme 2: Mechanism of conjugation between primary amine and succinimidyl carbonate
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Determining the Maximum Reagent Concentration for Effective Aptamer PEGylation
Six reaction mixtures, all containing a solvent system of one half acetonitrile, one half borate buffered saline
(BBS) solution (10 mM Na2B4O7, 150 mM NaCl, pH 9) were prepared. Their reagent concentations are as
follows:
1. 13.3 μM ZMYK-23 (5’ FAM, 3’ amino) and 12 mM mPEG-SC 20K.
2. 21.5 μM ZMYK-23 (5’ FAM, 3’ amino) and 19 μM mPEG-SC 20K.
3. 31.1 μM ZMYK-23 (5’ FAM, 3’ amino) and 28 mM mPEG-SC 20K.
4. 40.0 μM ZMYK-23 (5’ FAM, 3’ amino) and 36 mM mPEG-SC 20K.
5. 56.0 μM ZMYK-23 (5’ FAM, 3’ amino) and 50 mM mPEG-SC 20K.
6. 93.3 μM ZMYK-23 (5’ FAM, 3’ amino) and 83 mM mPEG-SC 20K.
They were each incubated for one hour at ambient temperature, then diluted to a final aptamer
concentration of 200 nM and characterized using agarose gel electrophoresis. The gel was 1.5% agarose in
0.5X TAE buffer and was run at 200 V for 20 minutes. The gel image is annotated in figure 16 in the
appendix.
Scheme 2: Mechanism of conjugation between primary amine and succinimidyl carbonate
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Isolation of PEGylated ZMYK-23 Through Electroelution
The reaction mixture was a solution of 40 μM ZMYK-23 (5’ FAM, 3’ amino) and 2 mM mPEG-SC 20K in
a solvent system of one half acetonitrile, one half borate buffered saline (BBS) solution (10 mM Na2B4O7,
150 mM NaCl, pH 9). It was incubated at 40°C for 90 minutes.
50 µL of the reaction mixture was loaded onto a 1.5% agarose gel in 0.5X TAE buffer and run at 150V for
45 minutes. The slice of gel containing the PEGylated aptamer was excised and placed into a D-TubeTM
Dialyser Midi MWCO 6-8 kDa tube along with 700 µL of 0.5X TAE buffer. Electroelution was performed
in a n electrophoresis developing tank at 150V for 25 minutes, then the polarity was reversed and it was run
at 125V for another 2 minutes.
The solution collected from electroelution was concentrated using a VWR® Centrifugal Filters (modified
PES, 30K, 500 µL). The solution was placed in a VWR® filter, which was then centrifuged for 10 minutes
at 3800 rcf. 50 µL DPBS was placed on the top of the column, allowed to incubate at ambient temperature
for 5 minutes, then collected.
The concentrated, purified PEG-ZMYK-23 solution was characterized using agarose gel electrophoresis. 2
µL of the solution was diluted to 10 µL with ddH2O and was loaded onto a 1.5% agarose gel in 0.5X TAE
buffer. 10 µL 200 nM FAM-labelled DNA library was loaded into another well as a control. The gel was
run at 200 V for 20 minutes. The gel image is annotated in figure 17 in the appendix.
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Viral Infectivity Assays
Titring Neutralizing Antibodies
Two 12-well plates containing ~95% confluent Vero cells in DMEM+10% FBS were prepared.
A series of solutions containing VSV∆51-YFP and polyclonal anti-VSV neutralizing antibodies (nAbs) in
DMEM were prepared. All solutions contained 400 pfu/mL VSV∆51-YFP, as well as various dilutions of
nAbs. The nAbs dilutions were as follows: 1/10000, 1/7500, 1/5000, 1/2500, 1/1250, 1/625, and
1/312.5, and 0 (no nAbs). The VSV∆51-YFP/nAbs solutions were each incubated at 37°C for one hour.
The medium was removed from the Vero cell-containing 12-well plates. 250 µL of each VSV∆51-
YFP/nAbs solution was added to each of 3 wells. The plates were then incubated at 37°C, 5% CO2 for one
hour.
An agarose overlay solution was prepared as follows: 15 mL of 1% agarose in ddH2O was heated until
homogenous. 15 mL 2X DMEM+10% FBS was added to the agarose solution. 1 mL of the solution was
gently placed on top of each well of the two 12-well plates. The plates were then incubated at 37°C, 5%
CO2 for 24 hours. Images of the plates were taken using the Fluorochem® Q from Alpha Innotech, and are
annotated in figure 18 in the appendix.
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Viral Infectivity Assay – Verifying Aptamer Pool’s Ability to Shield VSV from nAbs in vitro
Three 12-well plates containing ~95% confluent Vero cells in DMEM+10% FBS were prepared.
A 100 µM polyclonal aptamer pool containing equimolar amounts of the following VSV-binding aptamers
ZMYK-20, ZMYK-23, ZMYK-29, MS-50, SS-31, and SS-39 was prepared.
A series of solutions containing VSV∆51-YFP and aptamers were prepared. First, 30 µL 1.04e4 pfu/mL
VSV∆51-YFP was placed into each of 10 tubes (312 pfu per tube). Then, varying amounts of VSV-binding
aptamer was added to each tube as follows:
1. No aptamer
2. 8 µL 100 µM polyclonal aptamer pool (0.8 nmol total aptamer)
3. 20 µL 100 µM polyclonal aptamer pool (2.0 nmol total aptamer)
4. 30 µL 100 µM polyclonal aptamer pool (3.0 nmol total aptamer)
5. 60 µL 100 µM polyclonal aptamer pool (6.0 nmol total aptamer)
6. No aptamer
7. 8 µL 100 µM unlabelled ZMYK-23 (0.8 nmol total aptamer)
8. 20 µL 100 µM unlabelled ZMYK-23 (2.0 nmol total aptamer)
9. 30 µL 100 µM unlabelled ZMYK-23 (3.0 nmol total aptamer)
10. 60 µL 100 µM unlabelled ZMYK-23 (6.0 nmol total aptamer)
The tubes were incubated at 37°C for one hour. Following incubation, 30 µL of a 1/80 nAbs dilution in
DMEM was added to each of the 10 tubes, and they were again incubated at 37°C, 5% CO2 for one hour.
DMEM was added to the tubes such that each tube contained a total of 800 µL of liquid, then the tubes
were again incubated at 37°C, 5% CO2 for one hour.
The medium was removed from the Vero cell-containing 12-well plates. 250 µL of each VSV∆51-
YFP/aptamer/nAbs solution was added to each of 3 wells. The plates were then incubated at 37°C, 5%
CO2 for one hour.
An agarose overlay solution was prepared as follows: 15 mL of 1% agarose in ddH2O was heated until
homogenous. 15 mL 2X DMEM+10% FBS was added to the agarose solution. 1 mL of the solution was
gently placed on top of each well of the two 12-well plates. The plates were then incubated at 37°C, 5%
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CO2 for 24 hours. Images of the plates were taken using the Fluorochem® Q from Alpha Innotech, and are
annotated in figure 19 in the appendix.
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Flow Cytometric Competitive Binding Assay
A solution containing equimolar amounts of the 5’ FAM-labelled VSV-binding aptamers MS-50, SS-31, SS-
37, and SS-39, with a total aptamer concentration of 400 nM was prepared in DPBS.
Four tubes, each containing 2e9 pfu VSV∆51-YFP and 0.1 mg/mL yeast RNA in DPBS were prepared and
incubated at ambient temperature for 30 minutes. The tubes were labelled A-, A+, B-, and B+. The tubes
were centrifuged at 17200 rcf for 20 minutes, then the pellets were isolated.
50 µL of the 400 nM polyclonal VSV-binding aptamer solution was added to tubes A+ and B+. 50 µL
DPBS was added to A- and B-. The four tubes were again incubated at ambient temperature for 30 minutes,
centrifuged at 17200 rcf for 20 minutes, and the pellets were isolated.
The pellets of tubes B+ and B- were resuspended in 50 µL 2 mg/mL nAbs in DPBS and incubated at 37°C
for one hour. Tubes A+ and A- were resuspended in 300 µL DPBS and analysed with flow cytometry while
tubes B+ and B- were incubating with nAbs.
Following incubation of tubes B+ and B-, they were centrifuged at 17200 rcf for 20 minutes, then their
pellets were isolated and resuspended in 300 µL DPBS, and they were analysed with flow cytometry.
Histograms of the flow cytometric data compiled were analyzed using Kaluza® Flow Analysis Software from
Beckman Coulter and are displayed as figure 20 in the appendix.
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Vero Cell SELEX
Background
Aptamer research is still in its youth, and a wealth of new potential aptamer targets have yet to be explored.
In this project aptamer pools were selected for Vero cells, a novel target, through cell-SELEX.
Vero cells are one of the most widely used cell lines in research. Originally derived from the kidney
epithelial cells of an African green monkey (Cercopithecus aethiops), Vero cells are an anchorage-dependent,
continuous cell line. They have been invaluable in virology, parasitology, and oncology research since the
1960s 23. In Berezovski research group, Vero cells are an essential component of in vitro research into
oncolytic viruses.
The discovery of Vero cell-binding aptamers will serve as a proof of concept and will have many potential
benefits. One application of aptamers with high binding affinity to Vero cells is use in Aptamer-facilitated
BIomarker Discovery (AptaBID). In AptaBID, aptamers with high affinity and specificity to a particular cell
line are selected and used to discover cell surface biomarkers unique to the cell line 24. Cell surface
biomarkers are proteins that are expressed on the surfaces of some cells, and can be indicative of a cell’s
type, susceptibility to disease, or disease state. In recent years their use has grown in popularity, as they
have proven themselves to be invaluable tools in diagnostic medicine, with applications such as cancer
diagnosis and prognosis determination 25. AptaBID is accomplished by first selecting aptamers with the
desired binding characteristics, then isolating the aptamer-bound biomarker by lysing cells, and finally
analyzing the biomarker with mass spectroscopy to determine its structure 24.
Another application for Vero cell-binding aptamers is as a key component of a proof-of-concept experiment
dealing with enhanced VSV∆51 delivery currently planned in Berezovski research group. In the proposed
project, VSV∆51-binding aptamers and Vero cell-binding aptamers will be linked together via a bridging
oligonucleotide, which contains complementary sequences to both the virus-binding aptamer and the cell-
binding aptamer. Theoretically, by connecting the two aptamers, VSV∆51 will be able to reach its potential
host cells more rapidly and selectively, and will therefore be in circulation for a shorter amount of time, be
less likely to be neutralized, and be more potent. The development of Vero-cell binding aptamers is an
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essential component to this proof-of-concept project, and if the proof-of-concept is successful, similar
methods could one day be used clinically with human cancer cell-binding aptamers.
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Results & Discussion
Although this project was the shortest and the simplest of the three that make up my honours project, its
results were extremely promising. Unfortunately, due to time constraints, a full 8-12 rounds of SELEX plus
sequencing was not completed as hoped. Instead, five rounds of SELEX were performed and the resulting
oligonucleotide pools’ binding affinity to Vero cells was assessed through flow cytometry.
The procedure for the five rounds of selection that were performed were based on a protocol developed by
Shahrokh Ghobabloo, a fellow member of Berezovski research group. The rounds were successful,
producing adequate amounts of 100 nt oligonucleotide for analysis and subsequent analysis. Following the
five rounds of selection, oligonucleotide pools and a sample of Harvard DNA library were diluted to 25 nM
for use in flow cytometric analysis.
Results from the flow cytometric binding assay can be found in figure 21 in the appendix. The flow
cytometric histogram clearly shows that the oligonucleotide pools obtained through SELEX have increased
binding affinity to Vero cells compared to DNA library. All five pools show good binding affinity to Vero
cells, interestingly with the pools from rounds 1 and 2 having the weakest binding of the 5 rounds, and
round 3’s pool having the strongest. It is expected that each subsequent pool would have superior binding
than its predecessor, but that was not the case in this experiment. Though this results is unexpected, it is
not terribly concerning because the difference in binding between round 3’s and round 5’s pools is very
modest, and a number a variables can influence flow cytometric results on a small scale. This unexpected
result could be the result of slight oligonucleotide concentration differences, or differences in the way each
flow cytometry sample was handled (extent of washing, incubation time, heat, etc.).
Although the difference in Vero cell affinity between the DNA library and the SELEX-produced
oligonucleotide pools is modest, it is a promising and exciting results. A greater shift would have been
unlikely for two reasons – only five rounds of SELEX were performed, and the concentration of the
oligonucleotide pools used was quite low. Unfortunately, time constraints made it unfeasible to amplify,
isolate, and purify the five oligonucleotide pools obtained for a second time to obtain a greater mass of
DNA. Therefore we worked at a low oligonucleotide concentration during the experiment with flow
cytometry.
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Conclusions
This experiment is only partially complete, but it holds a great deal of promise for the future. The next
steps for this project will be to repeat the flow cytometric binding assay, this time using oligonucleotide
pools with a DNA concentration greater than 25 nM. This will provide greater and more valid insight into
the binding characteristics of each pools. Following that, SELEX will be completed. Five more rounds of
Vero cell SELEX should ideally be performed. In addition, three rounds of negative selection, which
involved selecting for the oligonucleotides that do not bind to a different cell line, should ideally be
performed to ensure that the aptamers discovered are specific to Vero cells. The resulting aptamer pools
should eventually be sequenced, synthesized, and again have their Vero-cell binding capacity assessed
through flow cytometry.
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Materials & Methods
Primers and Harvard DNA library were obtained from Integrated DNA Technologies (Coralville, USA). The
Harvard DNA library consists of a mixture of DNA oligonucleotides, each composed of a 60 nucleotide
semi-random sequence flanked by constant 20 nucleotide sequences at each end that serve as primer
regions. The forward primer (5’-CTC CTC TGA CTG TAA CCA CG-3’) was labelled with Cy5 on its 5’
end, whereas the reverse primer (5’-GGC-TTC-TGG-ACT-ACC-TAT-GC-3’) was modified with a reversed
5’ phosphate group.
Original samples of Vero cells were obtained from Dr. John C. Bell’s laboratory at the Ottawa Hospital
Research Institute (Ottawa, Canada). The medium used for all cell cultures was Dulbecco’s Modified Eagle
Medium (DMEM) + 10% FBS (HyClone, Thermo Scientific, Waltham, USA), and the cultures were grown
in HeracellTM 150i CO2 incubator (Thermo Scientific, Waltham, USA). Vero cell cultures were grown and
maintained according to Ammerman et al., 2008 22 with the exception that the cultures were grown in 150
mm dishes from BD Biosciences (Franklin Lakes, USA) rather than 75 cm2 cell cultures flasks.
KAPA2G Robust Hotstart PCR Kit from Kapa Biosystems (Wilmingtom, USA) was used to amplify
aptamer pools. Gel electrophoresis experiments were performed in 1X modified TAE buffer (40 mM Tris -
Acetate 100 µM EDTA, pH 8.2), which had been prepared in-lab. The vapo.protect Mastercycler proS
from Eppendorf (Hamburg, Germany) was used as the thermocycler in all PCRs. UltraPureTM Agarose was
obtained from Invitrogen (Carlsbad, USA). GelRedTM for visualizing bands on agarose gels was obtained
from Biotium (Hayward, USA). Gels were visualized using the Fluorochem® Q from Alpha Innotech (Santa
Clara, USA).
Amicon Ultra Filters (3 kDa, 2 mL) for cell lysate concentration and Ultrafree-DA Centrifugal Filter Units
were obtained from EMD-Millipore (Billerica, USA). Lambda Exonuclease and buffer were obtained from
New England Biolabs (Ipswitch, USA).
Flow cytometric analysis was performed using the Beckman Coulter Cytomics FC500 and flow cytometric
data was analysed using Kaluza® Flow Analysis Software from Beckman Coulter (Mississauga, Canada).
Dulbecco’s phosphate buffered saline (DPBS) with low Mg2+ and low Ca2+ was obtained from HyClone
(Thermo Scientific, Waltham, USA).
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Procedure for Vero cell SELEX
1. Prepare cells: Vero cells are cultured in 150 mm plates in DMEM + 10% FBS and grown to 85-100%
confluence prior to selection.
2. In low Ca2+, low Mg2+ DPBS, prepare Harvard DNA library (2000 μL, 2 μM) for the first round of
selection, or DNA pool (1000 μL, target concentration 500 ng/mL) for subsequent rounds by mixing
and heating at 95°C for 10 minutes, then cooling on ice for 5 minutes. This ensures the formation of
folded ssDNA.
3. Remove the medium from the plate containing Vero cells and gently apply the DNA library or pool.
Shake at 200 rpm, 37°C for 5 minutes.
4. Remove the liquid from the plate and washed three times with low Ca2+, low Mg2+ DPBS.
5. Apply 1500 μL 10 mM EDTA in low Ca2+, low Mg2+ DPBS to the plate. Incubate at 37°C, 5% CO2
for 10 minutes.
6. Pipette the liquid up and down on the plate to thoroughly detach Vero cells from the surface of the
plate (this takes about 10 minutes). Collect the liquid suspension.
7. Heat the suspension at 95°C for 10 minutes, then centrifuge at 13,100 rcf for 5 minutes and collect
the supernatant.
8. Using an Amicon Ultra Filter (3k, 2 mL), concentrate the lysate to 50 μL.
9. Prepare PCR mixture with forward Cy5-labelled and reversed 5’-phosphate group primers as follows:
1X KAPA GC buffer with 1.5 mM MgCl2
500 nM each primer
500 μM dNTPs
0.02 UμL-1 KAPA2G Robust HotStart DNA Polymerase
1/50 dilution of concentrated cell lysate (from step 8)
10. Run the following PCR program:
2 minutes at 95°C
35 cycles of:
5 seconds at 95°C
20 seconds at 56°C
10 seconds at 72°C
Hold at 4°C
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11. Load 50 μL of the PCR solution into each well of a 3% agarose + 1X GelRedTM gel in 1X modified
TAE buffer. Run the gel at 150 V for 45 minutes.
12. Visualize bands on the gel under UV light using the Fluorochem® Q and excise the gel slices
containing the 100 nucleotide dsDNA fragments. Purify using Ultrafree-DA Centrifugal Filter Units.
13. Confirm the product by running 10 μL of the purified dsDNA on a 3% agarose gel in 1X modified
TAE at 150 V for 25 minutes
14. Digest the product with Lambda exonuclease according to the protocol provided by New England
Biolabs.
The preceding protocol was repeated five time, resulting in five aptamer pools for Vero cells. The binding
affinity of these pools to Vero cells was assessed using flow cytometry.
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Procedure for Flow Cytometric Binding Assay
A 150 mm plate containing 95% confluent Vero cells was trypsinized and resuspended in 10 mL DMEM +
10% FBS. 500,000 cells were plates into each of 7 1.5 mL tubes, which were then centrifuged at 500 rcf for
5 minutes. The supernatant was removed from each tubes, then the tubes were labelled A-G, and the
following was added to each:
A. 200 µL low Ca2+, low Mg2+ DPBS
B. 200 µL 25 nM Cy5-labelled Harvard DNA library in low Ca2+, low Mg2+ DPBS
C. 200 µL 25 nM Cy5-labelled round 1 Vero cell SELEX aptamer pool in low Ca2+, low Mg2+ DPBS
D. 200 µL 25 nM Cy5-labelled round 2 Vero cell SELEX aptamer pool in low Ca2+, low Mg2+ DPBS
E. 200 µL 25 nM Cy5-labelled round 3 Vero cell SELEX aptamer pool in low Ca2+, low Mg2+ DPBS
F. 200 µL 25 nM Cy5-labelled round 4 Vero cell SELEX aptamer pool in low Ca2+, low Mg2+ DPBS
G. 200 µL 25 nM Cy5-labelled round 5 Vero cell SELEX aptamer pool in low Ca2+, low Mg2+ DPBS
The tubes were incubated at 37°C for 5 minutes, then centrifuged at 500 rcf for 5 minutes. The
supernatants were removed, all pellets were resuspended in 200 µL low Ca2+, low Mg2+ DPBS, and
centrifuged at 500 rcf for 5 minutes. Once again, the supernatants were removed. Then each pellet was
resuspended in 500 µL low Ca2+, low Mg2+ DPBS and each suspension was analyzed using flow cytometry.
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References
1. Watson, James D., and Francis HC Crick. "Molecular structure of nucleic acids." Nature 171.4356
(1953): 737-738.
2. Tuerk, Craig, and Larry Gold. "Systematic evolution of ligands by exponential enrichment: RNA
ligands to bacteriophage T4 DNA polymerase." Science249.4968 (1990): 505-510.
3. Ellington, Andrew D., and Jack W. Szostak. "In vitro selection of RNA molecules that bind specific
ligands." Nature 346.6287 (1990): 818-822.
4. Jayasena, Sumedha D. "Aptamers: an emerging class of molecules that rival antibodies in
diagnostics." Clinical chemistry 45.9 (1999): 1628-1650.
5. Marimuthu, Citartan, et al. "Single-stranded DNA (ssDNA) production in DNA aptamer
generation." Analyst 137.6 (2012): 1307-1315.
6. Pierce, Kenneth E., et al. "Linear-After-The-Exponential (LATE)-PCR: primer design criteria for
high yields of specific single-stranded DNA and improved real-time detection." Proceedings of the
National Academy of Sciences of the United States of America 102.24 (2005): 8609-8614.
7. Rice, John E., et al. "Monoplex/multiplex linear-after-the-exponential-PCR assays combined with
PrimeSafe and Dilute-'N'-Go sequencing." Nature protocols 2.10 (2007): 2429-2438.
8. Sanchez, J. Aquiles, et al. "Linear-After-The-Exponential (LATE)–PCR: An advanced method of
asymmetric PCR and its uses in quantitative real-time analysis." Proceedings of the National Academy of
Sciences of the United States of America 101.7 (2004): 1933-1938.
9. Frey, Ulrich H., et al. "PCR-amplification of GC-rich regions:'slowdown PCR'."Nature
protocols 3.8 (2008): 1312-1317.
10. DeVita, Vincent T., and Edward Chu. "A history of cancer chemotherapy."Cancer research 68.21
(2008): 8643-8653.
11. Liu, T. C., and D. Kirn. "Gene therapy progress and prospects cancer: oncolytic viruses." Gene
therapy 15.12 (2008): 877-884.
12. Alajez, Nehad M., et al. "Enhanced vesicular stomatitis virus (VSVDelta51) targeting of head and
neck cancer in combination with radiation therapy or ZD6126 vascular disrupting agent." Cancer
Cell Int 12 (2012): 27.
51
Sele
cted
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ics
in A
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|
4/15
/20
14
13. Stojdl, David F., et al. "Exploiting tumor-specific defects in the interferon pathway with a
previously unknown oncolytic virus." Nature medicine 6.7 (2000): 821-825.
14. Muharemagic, Darija, et al. "Anti-fab aptamers for shielding virus from neutralizing
antibodies." Journal of the American Chemical Society 134.41 (2012): 17168-17177.
15. Labib, Mahmoud, et al. "Electrochemical sensing of aptamer-facilitated virus
immunoshielding." Analytical chemistry 84.3 (2012): 1677-1686.
16. Govan, Jeane M., Andrew L. McIver, and Alexander Deiters. "Stabilization and photochemical
regulation of antisense agents through PEGylation."Bioconjugate chemistry 22.10 (2011): 2136-2142.
17. Bailon, Pascal, and Chee-Youb Won. "PEG-modified biopharmaceuticals." (2009): 1-16.
18. Hamidi, Mehrdad, Amir Azadi, and Pedram Rafiei. "Pharmacokinetic consequences of
pegylation." Drug delivery 13.6 (2006): 399-409.
19. Jäschke, Andres, et al. "Synthesis and properties of oligodeoxyribonucleotide—polyethylene glycol
conjugates." Nucleic acids research 22.22 (1994): 4810-4817.
20. Kang, Hyungu, et al. "Characterization of PEGylated Anti-VEGF aptamers using surface plasmon
resonance." Macromolecular Research 16.2 (2008): 182-184.
21. Zassenhaus, H. Peter, Ronald A. Butow, and Yolanda P. Hannon. "Rapid electroelution of nucleic
acids from agarose and acrylamide gels." Analytical biochemistry 125.1 (1982): 125-130.
22. Diallo, Jean-Simon, et al. "Propagation, purification, and in vivo testing of oncolytic vesicular
stomatitis virus strains." Oncolytic Viruses. Humana Press, 2012. 127-140.
23. Ammerman, Nicole C., Magda Beier‐Sexton, and Abdu F. Azad. "Growth and maintenance of
Vero cell lines." Current protocols in microbiology (2008): A-4E.
24. Berezovski, Maxim V., et al. "Aptamer-facilitated biomarker discovery (AptaBiD)." Journal of the
American Chemical Society 130.28 (2008): 9137-9143.
25. Joensson, Haakan N., et al. "Detection and Analysis of Low‐Abundance Cell‐Surface Biomarkers
Using Enzymatic Amplification in Microfluidic Droplets."Angewandte Chemie International
Edition 48.14 (2009): 2518-2521.
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Appendix
Figures
FIGURE 2: AGAROSE GEL IMAGE OF LIMIT OF DETECTION ASSAY FOR SYMMETRIC-THEN-ASYMMETRIC PCR. 10 µL OF
EACH SYMMETRIC-THEN-ASYMMETRIC PCR MIXTURE WAS LOADED INTO A WELL. IN ADDITION, 5 µL MASSRULER LOW
RANGE DNA LADDER, READY-TO-USE FROM THERMO SCIENTIFIC WAS LOADED INTO THE CENTRE WELL. THE GEL WAS
3% AGAROSE IN 0.5X TAE BUFFER, AND WAS RUN AT 150 V FOR 45 MINUTES. THE GEL WAS VISUALIZED USING THE
FLUOROCHEM® Q FROM ALPHA INNOTECH ON THE CY2 FLUORESCENCE SETTING. FULL PROCEDURAL INFORMATION
CAN BE FOUND ON PAGES 11-12.
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FIGURE 3: AGAROSE GEL IMAGE OF FORWARD TO REVERSE PRIMER RATIO PLUS ANNEALING TEMPERATURE ASSAY, THE
FIRST EXPERIMENT OF LATE-PCR PROTOCOL OPTIMIZATION. FOLLOWING PCR, 10 µL OF EACH SAMPLE WAS LOADED
INTO A WELL, GROUPED ACCORDING TO THEIR PRIMER RATIOS. NOTE THAT A NEGATIVE CONTROL (I.E. NO TEMPLATE
DNA) FOR EACH PRIMER RATIO WAS PERFORMED, AND IS HERE DENOTED BY A NEGATIVE SYMBOL (-). IN ADDITION, 5
µL MASSRULER LOW RANGE DNA LADDER, READY-TO-USE FROM THERMO SCIENTIFIC WAS LOADED INTO A WELL.
THE GEL WAS 3% AGAROSE IN 0.5X TAE BUFFER, AND WAS RUN AT 150 V FOR 45 MINUTES. THE GEL WAS VISUALIZED
USING THE FLUOROCHEM® Q FROM ALPHA INNOTECH ON THE CY3 FLUORESCENCE SETTING. FULL PROCEDURAL
INFORMATION CAN BE FOUND ON PAGE 13.
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FIGURE 4: AGAROSE GEL IMAGE OF LATE-PCR CYCLE NUMBER PLUS ANNEALING TEMPERATURE ASSAY, THE SECOND
EXPERIMENT OF LATE-PCR PROTOCOL OPTIMIZATION. EACH PCR SAMPLE INITIALLY CONTAINED 17 NM TEMPLATE
DNA. FOLLOWING PCR, 10 µL OF EACH SAMPLE WAS LOADED INTO A WELL, GROUPED ACCORDING TO THE NUMBER
OF PCR CYCLES THEY HAD BEEN SUBJECT TO. THE GEL WAS 3% AGAROSE IN 0.5X TAE BUFFER, AND WAS RUN AT 150
V FOR 55 MINUTES. THE GEL WAS VISUALIZED USING THE FLUOROCHEM® Q FROM ALPHA INNOTECH ON THE CY2
FLUORESCENCE SETTING. FULL PROCEDURAL INFORMATION CAN BE FOUND ON PAGE 14.
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FIGURE 5: AGAROSE GEL IMAGE OF LATE-PCR CYCLE NUMBER PLUS ANNEALING TEMPERATURE NEGATIVE CONTROLS.
NONE OF THE PCR SAMPLES CONTAINED ANY TEMPLATE DNA. FOLLOWING PCR, 10 µL OF EACH SAMPLE WAS
LOADED INTO A WELL, GROUPED ACCORDING TO THE NUMBER OF PCR CYCLES THEY HAD BEEN SUBJECT TO. THE GEL
WAS 3% AGAROSE IN 0.5X TAE BUFFER, AND WAS RUN AT 150 V FOR 55 MINUTES. THE GEL WAS VISUALIZED USING
THE FLUOROCHEM® Q FROM ALPHA INNOTECH ON THE CY2 FLUORESCENCE SETTING. FULL PROCEDURAL
INFORMATION CAN BE FOUND ON PAGE 14.
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FIGURE 6: AGAROSE GEL IMAGE COMPARING PCR PRODUCTS AND NEGATIVE CONTROLS OF LATE-PCRS PERFORMED
USING A TRUNCATED (17 NT) FORWARD PRIMER AND A FULL-LENGTH (20 NT) FORWARD PRIMER. FOLLOWING PCR,
10 µL OF EACH PCR PRODUCT WAS LOADED INTO A WELL. IN ADDITION, 10 µL 200 NM N-40 DNA LIBRARY WAS
LOADED INTO THE CENTRE WELL. THE GEL WAS 3% AGAROSE IN 0.5X TAE BUFFER, AND WAS RUN AT 150 V FOR 25
MINUTES. THE GEL WAS VISUALIZED USING THE FLUOROCHEM® Q FROM ALPHA INNOTECH ON THE CY2
FLUORESCENCE SETTING. FULL PROCEDURAL INFORMATION CAN BE FOUND ON PAGE 15.
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FIGURE 7: AGAROSE GEL IMAGE OF LIMIT OF DETECTION ASSAY FOR LATE-PCR AND SYMMETRIC-THEN-ASYMMETRIC
PCR. FOLLOWING PCR, 10 µL OF EACH PCR PRODUCT WAS LOADED INTO A WELL. IN ADDITION, 10 µL 200 NM N-40 DNA LIBRARY WAS LOADED INTO THE CENTRE WELL. THE GEL WAS 3% AGAROSE IN 0.5X TAE BUFFER, AND WAS
RUN AT 150 V FOR 50 MINUTES. THE GEL WAS VISUALIZED USING THE FLUOROCHEM® Q FROM ALPHA INNOTECH ON
THE CY2 FLUORESCENCE SETTING. FULL PROCEDURAL INFORMATION CAN BE FOUND ON PAGES 16-18.
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FIGURE 8: AGAROSE GEL IMAGE OF LATE-PCR PRODUCTS MADE USING KAPA2G ROBUST HOTSTART DNA
POLYMERASE. BOTH A NEGATIVE CONTROL PCR MIXTURE (WITH NO TEMPLATE DNA) AND AN EXPERIMENTAL
MIXTURE (WITH 170 PM N-40 DNA LIBRARY TEMPLATE) WERE PREPARED AND ANALYZED. FOLLOWING PCR, 10 µL OF
EACH PCR PRODUCT WAS LOADED INTO A WELL. IN ADDITION, 10 µL 200 NM N-40 DNA LIBRARY WAS LOADED INTO
THE CENTRE WELL. THE GEL WAS 3% AGAROSE IN 0.5X TAE BUFFER, AND WAS RUN AT 150 V FOR 30 MINUTES. THE
GEL WAS VISUALIZED USING THE FLUOROCHEM® Q FROM ALPHA INNOTECH ON THE CY2 FLUORESCENCE SETTING.
FULL PROCEDURAL INFORMATION CAN BE FOUND ON PAGE 19.
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FIGURE 9: AGAROSE GEL IMAGE ASSESSING THE EFFECT OF REACTION TIME ON PEGYLATION YIELD OF A REACTION
MIXTURE CONTAINING THE 79 NUCLEOTIDE SSDNA APTAMER ZMYK-23 (5’ FAM, 3’ AMINO) AND 20 KDA MPEG-SC.
5 µL OF THE REACTION MIXTURE (TOTAL APTAMER CONCENTRATION 2 µM) WAS LOADED INTO THE GEL, ALONG WITH
25 µL 200 NM N40 DNA LIBRARY (80 NUCLEOTIDE SSDNA). THE GEL WAS 1.5% AGAROSE IN 0.5X TAE BUFFER, AND
WAS RUN AT 150 V FOR 30 MINUTES. THE GEL WAS VISUALIZED USING THE FLUOROCHEM® Q FROM ALPHA INNOTECH
ON THE CY2 FLUORESCENCE SETTING. FULL PROCEDURAL INFORMATION CAN BE FOUND ON PAGE 28.
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FIGURE 10: AGAROSE GEL IMAGE ASSESSING THE EFFECTIVENESS OF A ½ SODIUM CARBONATE (PH 11), ½
ACETONITRILE REACTION MIXTURE BUFFER SOLUTION ON SUPPORTING PEGYLATION OF A REACTION MIXTURE
CONTAINING THE 79 NUCLEOTIDE SSDNA APTAMER ZMYK-23 (5’ FAM, 3’ AMINO) AND 20 KDA MPEG-SC. 5 µL OF
THE REACTION MIXTURE (TOTAL APTAMER CONCENTRATION 1 µM) WAS LOADED INTO THE GEL, ALONG WITH 25 µL
200 NM N-40 DNA LIBRARY (80 NUCLEOTIDE SSDNA). THE GEL WAS 1.5% AGAROSE IN 0.5X TAE BUFFER, AND WAS
RUN AT 150 V FOR 25 MINUTES. THE GEL WAS VISUALIZED USING THE FLUOROCHEM® Q FROM ALPHA INNOTECH ON
THE CY2 FLUORESCENCE SETTING. FULL PROCEDURAL INFORMATION CAN BE FOUND ON PAGE 29.
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FIGURE 11: AGAROSE GEL IMAGE ASSESSING THE EFFECTIVENESS OF A ½ BORATE BUFFERED SALINE (PH 9), ½
ACETONITRILE REACTION MIXTURE BUFFER SOLUTION ON SUPPORTING PEGYLATION OF A REACTION MIXTURE
CONTAINING THE 79 NUCLEOTIDE SSDNA APTAMER ZMYK-23 (5’ FAM, 3’ AMINO) AND 20 KDA MPEG-SC. 5 µL OF
THE REACTION MIXTURE (TOTAL APTAMER CONCENTRATION 1 µM) WAS LOADED INTO THE GEL, ALONG WITH 25 µL
200 NM N-40 DNA LIBRARY (80 NUCLEOTIDE SSDNA). THE GEL WAS 1.5% AGAROSE IN 0.5X TAE BUFFER, AND WAS
RUN AT 200 V FOR 25 MINUTES. THE GEL WAS VISUALIZED USING THE FLUOROCHEM® Q FROM ALPHA INNOTECH ON
THE CY2 FLUORESCENCE SETTING. FULL PROCEDURAL INFORMATION CAN BE FOUND ON PAGE 30.
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FIGURE 12: AGAROSE GEL IMAGE ASSESSING THE EFFECT OF VARYING THE CONCENTRATION OF ACETONITRILE ON
APTAMER PEGYLATION IN A REACTION MIXTURE CONTAINING THE 79 NUCLEOTIDE SSDNA APTAMER ZMYK-23 (5’
FAM, 3’ AMINO) AND 20 KDA MPEG-SC IN AN ACETONITRILE AND BORATE BUFFERED SALINE (PH 9) BUFFER
SOLUTION. 5 µL OF EACH REACTION MIXTURE (TOTAL APTAMER CONCENTRATION 1 µM) WAS LOADED INTO THE GEL,
ALONG WITH 25 µL 200 NM N-40 DNA LIBRARY (80 NUCLEOTIDE SSDNA). THE GEL WAS 1.5% AGAROSE IN 0.5X
TAE BUFFER, AND WAS RUN AT 200 V FOR 25 MINUTES. THE GEL WAS VISUALIZED USING THE FLUOROCHEM® Q FROM
ALPHA INNOTECH ON THE CY2 FLUORESCENCE SETTING. FULL PROCEDURAL INFORMATION CAN BE FOUND ON PAGE
31.
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FIGURE 13: AGAROSE GEL IMAGE ASSESSING THE EFFECT OF HEAT AND AGITATION ON APTAMER PEGYLATION IN A
REACTION MIXTURE CONTAINING THE 79 NUCLEOTIDE SSDNA APTAMER ZMYK-23 (5’ FAM, 3’ AMINO) AND 20 KDA
MPEG-SC IN A ½ ACETONITRILE AND ½ BORATE BUFFERED SALINE (PH 9) BUFFER SOLUTION. THE REACTION MIXTURE
WAS INCUBATED AT 50°C, 300 RPM FOR ONE HOUR. FOLLOWING INCUBATION, 5 µL OF THE REACTION MIXTURE
(TOTAL APTAMER CONCENTRATION 1 µM) WAS LOADED INTO THE GEL, ALONG WITH 25 µL 200 NM N-40 DNA
LIBRARY (80 NUCLEOTIDE SSDNA). THE GEL WAS 1.5% AGAROSE IN 0.5X TAE BUFFER, AND WAS RUN AT 200 V FOR
20 MINUTES. THE GEL WAS VISUALIZED USING THE FLUOROCHEM® Q FROM ALPHA INNOTECH ON THE CY2
FLUORESCENCE SETTING. FULL PROCEDURAL INFORMATION CAN BE FOUND ON PAGE 32.
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FIGURE 14: AGAROSE GEL IMAGE ASSESSING THE EFFECT OF VARYING THE ORGANIC SOLVENT USED IN THE REACTION
BUFFER SOLUTION ON APTAMER PEGYLATION. THE REACTION MIXTURES CONTAINED THE 79 NUCLEOTIDE SSDNA
APTAMER ZMYK-23 (5’ FAM, 3’ AMINO) AND 20 KDA MPEG-SC IN A ½ BORATE BUFFERED SALINE (PH 9), ½
ORGANIC SOLVENT BUFFER SOLUTION. 5 µL OF EACH REACTION MIXTURE (TOTAL APTAMER CONCENTRATION 1 µM)
WAS LOADED INTO THE GEL, ALONG WITH 25 µL 200 NM N-40 DNA LIBRARY (80 NUCLEOTIDE SSDNA). THE GEL WAS
1.5% AGAROSE IN 0.5X TAE BUFFER, AND WAS RUN AT 200 V FOR 30 MINUTES. THE GEL WAS VISUALIZED USING THE
FLUOROCHEM® Q FROM ALPHA INNOTECH ON THE CY2 FLUORESCENCE SETTING. FULL PROCEDURAL INFORMATION
CAN BE FOUND ON PAGE 33.
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FIGURE 15: AGAROSE GEL IMAGES ASSESSING THE EFFECT OF REAGENT CONCENTRATION ON APTAMER PEGYLATION IN
REACTION MIXTURES CONTAINING THE 79 NUCLEOTIDE SSDNA APTAMER ZMYK-23 (5’ FAM, 3’ AMINO) AND 20 KDA
MPEG-SC IN ½ ACETONITRILE AND ½ BORATE BUFFERED SALINE (PH 9) BUFFER SOLUTIONS. EACH REACTION MIXTURE
CONTAINED A 100 X MOLAR EXCESS OF MPEG-SC OVER ZMYK-23. EACH REACTION MIXTURE WAS DILUTED TO AN
APTAMER CONCENTRATION OF 200 NM IN DDH2O, THEN 25 µL OF EACH DILUTE REACTION MIXTURE WAS LOADED
INTO THE GEL, ALONG WITH 25 µL 200 NM N-40 DNA LIBRARY (80 NUCLEOTIDE SSDNA). THE GEL WAS 1.5%
AGAROSE IN 0.5X TAE BUFFER, AND WAS RUN AT 200 V FOR 20 MINUTES. THE GEL WAS VISUALIZED USING THE
FLUOROCHEM® Q FROM ALPHA INNOTECH ON THE CY2 FLUORESCENCE SETTING. FULL PROCEDURAL INFORMATION
CAN BE FOUND ON PAGE 34.
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FIGURE 16: AGAROSE GEL IMAGE ASSESSING THE EFFECT OF REAGENT CONCENTRATION ON APTAMER PEGYLATION IN A
REACTION MIXTURE CONTAINING THE 79 NUCLEOTIDE SSDNA APTAMER ZMYK-23 (5’ FAM, 3’ AMINO) AND 20 KDA
MPEG-SC IN ½ ACETONITRILE AND ½ BORATE BUFFERED SALINE (PH 9) BUFFER SOLUTIONS. EACH REACTION MIXTURE
CONTAINED A 900 X MOLAR EXCESS OF MPEG-SC OVER ZMYK-23. EACH REACTION MIXTURE WAS DILUTED TO AN
APTAMER CONCENTRATION OF 200 NM IN DDH2O, THEN 10 µL OF EACH DILUTE REACTION MIXTURE WAS LOADED
INTO THE GEL, ALONG WITH 10 µL 200 NM N-40 DNA LIBRARY (80 NUCLEOTIDE SSDNA). THE GEL WAS 1.5%
AGAROSE IN 0.5X TAE BUFFER, AND WAS RUN AT 200 V FOR 20 MINUTES. THE GEL WAS VISUALIZED USING THE
FLUOROCHEM® Q FROM ALPHA INNOTECH ON THE CY2 FLUORESCENCE SETTING. FULL PROCEDURAL INFORMATION
CAN BE FOUND ON PAGE 35.
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FIGURE 17: AGAROSE GEL IMAGE OF PEGYLATED ZMYK-23 (5’ FAM) PURIFIED BY ELECTROELUTION. 10 µL OF THE
PURIFIED APTAMER WAS LOADED INTO THE GEL, ALONG WITH 10 µL 200 NM N-40 DNA LIBRARY (80 NUCLEOTIDE
SSDNA). THE GEL WAS 1.5% AGAROSE IN 0.5X TAE BUFFER, AND WAS RUN AT 200 V FOR 20 MINUTES. THE GEL WAS
VISUALIZED USING THE FLUOROCHEM® Q FROM ALPHA INNOTECH ON THE CY2 FLUORESCENCE SETTING. FULL
PROCEDURAL INFORMATION CAN BE FOUND ON PAGE 36.
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← No nAbs ← 1/2500
← 1/10000 ← 1/1250
← 1/7500 ← 1/625
← 1/5000 ← 1/312
FIGURE 18: IMAGES OF 12-WELL PLATES USED IN ANTI-VSV∆51 NABS T ITERING EXPERIMENT. EACH WELL CONTAINS
95% VERO CELLS, ALONG WITH 100 PFU VSV∆51 THAT HAD PREVIOUSLY BEEN INCUBATED WITH A PARTICULAR
DILUTIONS OF ANTI-VSV∆51 NABS. THE PLATES WERE VISUALIZED USING THE FLUOROCHEM® Q FROM ALPHA
INNOTECH ON THE CY2 FLUORESCENCE SETTING. FULL PROCEDURAL INFORMATION CAN BE FOUND ON PAGE 37.
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A A B C D E F G H I J K
FIGURE 19: IMAGES OF 12-WELL PLATES USED IN A VIRAL INFECTIVITY ASSESSING THE ABILITY OF VSV∆51-BINDING
APTAMERS TO PROTECT VSV∆51 FROM NABS. EACH WELL CONTAINS 95% VERO CELLS, ALONG WITH 100 PFU
VSV∆51 THAT HAD PREVIOUSLY BEEN INCUBATED WITH A PARTICULAR MIXTURE AND CONCENTRATION OF VSV∆51-BINDING APTAMERS, FOLLOWED BY ANTI-VSV∆51 NABS. THE CONTENTS OF CONTENTS OF EACH TRIPLICATE GROUP
OF WELLS ARE LISTED BELOW.
A: NEGATIVE CONTROL (NO VSV∆51-YFP, NABS, OR APTAMERS) B: POSITIVE CONTROL (100 PFU VSV∆51-YFP PER WELL) C: VSV∆51-YFP (100 PFU PER WELL), NABS D: VSV∆51-YFP (100 PFU PER WELL), NABS, POLYCLONAL VSV-BINDING APTAMER MIXTURE (0.25 NMOL PER WELL) E: VSV∆51-YFP (100 PFU PER WELL), NABS, POLYCLONAL VSV-BINDING APTAMER MIXTURE (0.625 NMOL PER WELL) F: VSV∆51-YFP (100 PFU PER WELL), NABS, POLYCLONAL VSV-BINDING APTAMER MIXTURE (0.9375 NMOL PER WELL) G: VSV∆51-YFP (100 PFU PER WELL), NABS, POLYCLONAL VSV-BINDING APTAMER MIXTURE (0.1875 NMOL PER WELL) H: VSV∆51-YFP (100 PFU PER WELL), NABS, MONOCLONAL ZMYK-23 (0.25 NMOL PER WELL) I: VSV∆51-YFP (100 PFU PER WELL), NABS, MONOCLONAL ZMYK-23 (0.625 NMOL PER WELL) J: VSV∆51-YFP (100 PFU PER WELL), NABS, MONOCLONAL ZMYK-23 (0.9375 NMOL PER WELL) K: VSV∆51-YFP (100 PFU PER WELL), NABS, MONOCLONAL ZMYK-23 (1.875 NMOL PER WELL)
THE PLATES WERE VISUALIZED USING THE FLUOROCHEM® Q FROM ALPHA INNOTECH ON THE CY2 FLUORESCENCE
SETTING. FULL PROCEDURAL INFORMATION CAN BE FOUND ON PAGE 38-39.
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Fluorescence Intensity
FIGURE 20: HISTOGRAM OF FLOW CYTOMETRIC DISPLACEMENT ASSAY DATA MEASURING THE ABILITY OF ANTI-VSV∆51
NABS PRODUCED BY BUNNY #2 TO DISPLACE A POLYCLONAL MIXTURE OF VSV∆51-BINDING APTAMERS FROM THE
SURFACE OF VSV∆51. FLOW CYTOMETRY WAS PERFORMED WITH THE BECKMAN COULTER CYTOMICS FC500 AND
FLOW CYTOMETRIC DATA WAS ANALYSED USING KALUZA® FLOW ANALYSIS SOFTWARE FROM BECKMAN COULTER.
FULL PROCEDURAL DATA CAN BE FOUND OF PAGE 40.
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Fluorescence Intensity
FIGURE 21: HISTOGRAM OF FLOW CYTOMETRIC BINDING ASSAY MEASURING THE BINDING OF CY5-LABELLED
OLIGONUCLEOTIDE POOLS SELECTED THROUGH VERO CELL SELEX AND OF HARVARD DNA LIBRARY TO BIND TO VERO
CELLS. EACH SAMPLE CONTAINED A TOTAL OF 500,000 VERO CELLS AND 5 NMOL OLIGONUCLEOTIDE. FLOW
CYTOMETRY WAS PERFORMED WITH THE BECKMAN COULTER CYTOMICS FC500 AND FLOW CYTOMETRIC DATA WAS
ANALYSED USING KALUZA® FLOW ANALYSIS SOFTWARE FROM BECKMAN COULTER. FULL PROCEDURAL DATA CAN BE
FOUND OF PAGE 48.
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Protocol – LATE-PCR for Single Stranded Oligonucleotide Pool
Amplification
Apparatus and Materials
PCR reaction mixture containers
0.2 mL PCR tubes
Thermocycler
Eppendorf Mastercycler® pro S
LATE-PCR using Taq Polymerase
Reagents and Chemicals
Distilled, deionized water (ddH2O)
Gotaq® Hot Start DNA Polymerase (Promega)
5X Green GoTaq® Flexi Buffer
Reverse DNA Primer: (5’-GGC TTC TGG ACT ACC TAT GC-3’)
5' FAM labelled Forward DNA Primer (5’-CTC CTC TGA CTG TAA CCA CG-3’)
dNTP mix (10 mM stock)
Protocol
Prepare a master mix containing the following components at the following concentrations:
1X Green GoTaq® Flexi Buffer (Promega), from 5X concentration stock
2.5 mM MgCl2 (Promega)
25 nM reverse DNA primer (5'-GGC TTC TGG ACT ACC TAT GC-3')
1 uM 5' FAM-labelled forward primer (5'-CTC CTC TGA CTG TAA CCA CG-3')
400 uM dNTPs
0.025 UuL-1 Gotaq® Hot Start DNA Polymerase (Promega)
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Combine this master mix with the oligonucleotide pool with a ratio of 1 volume oligonucleotide pool per 5
volumes master mix.
Use the following thermocycler program to amplify the oligonucleotide pools:
Initial denaturation: 2 minutes at 95º C
Followed by 50 cycles of the following steps:
Denaturation: 10 seconds at 95º C
Annealing: 20 seconds at 56º C
Extension: 10 seconds at 72º C
Hold at 4º C
LATE-PCR using KAPA DNA Polymerase
For more difficult amplifications, including those involving high GC content
Reagents and Chemicals
Distilled, deionized water (ddH2O)
KAPA2G Robust Hot Start DNA Polymerase (KAPA Biosystems)
5X KAPA2G Buffer A (KAPA Biosystems)
Reverse DNA Primer (5’-GGC TTC TGG ACT ACC TAT GC-3’)
5' FAM labelled Forward DNA Primer (5’-CTC CTC TGA CTG TAA CCA CG-3’)
dNTP mix (10 mM stock)
Protocol
Prepare a master mix containing the following components at the following concentrations:
1X KAPA2G Buffer A, from 5X concentration stock
25 nM reverse DNA primer (5'-GGC TTC TGG ACT ACC TAT GC-3')
1 uM 5' FAM-labelled forward primer (5'-CTC CTC TGA CTG TAA CCA CG-3')
400 uM dNTPs
0.025 UuL-1 KAPA2G Robust Hot Start DNA Polymerase
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Combine this master mix with the oligonucleotide pool with a ratio of 1 volume oligonucleotide pool per 5
volumes master mix.
Use the following thermocycler program to amplify the oligonucleotide pools:
Initial denaturation: 2 minutes at 95º C
Followed by 40 cycles of the following steps:
Denaturation: 10 seconds at 95º C
Annealing: 20 seconds at 56º C
Extension: 10 seconds at 72º C
Hold at 4º C