Dugger FGF8-b

Aptamer Selection against Mammalian Fibroblast Growth Factor 8 b for Early Detection of

Prostate Cancer

Samuel Dugger

October 8, 2013

Fall 2013

N71 RNA Pool

Talon Bead-based Selection

Mammalian Fibroblast Growth Factor 8 b Isoform

Dugger FGF8-b

Abstract:

Despite major advances in oncology over the past few years, cancer still remains one of the most

prevalent and deadly diseases affecting mankind today. Even in developed countries like the United

States, cancer is one of the top causes of death besides heart disease (Jemal et al, 2011). Although the

word “cancer” is used to describe a family of diseases rather than a specific ailment, all cancers are

identified by unregulated cell division that results in the formation of tumors that may spread throughout

the body (Croce, 2008). Recent studies have suggested that fibroblast growth factors (FGFs) may

contribute to the growth of cancers. Overexpressed FGF8b in particular has been linked with prostate

cancer (Kwabi-Addo, 2004).

The main objective of this research is to find an aptamer that could detect FGF8b. Aptamers are

oligonucleotides with a high binding affinity for certain molecules, and they have a variety of applications

in areas such as drug delivery, diagnostics, therapeutics, developmental biology, and systems and

synthetic biology. They have also been proven to be more accurate, more durable, and cheaper to produce

than antibodies (which are often used for similar reasons). No custom antibodies have been successfully

produced that can locate this target, and aptamers could prove to be the solution.

Successfully isolating and amplifying an aptamer that can locate FGF8b could help doctors detect

prostate cancer in earlier stages and vastly improve patient survival rates. Additionally, this aptamer could

be used for targeted drug delivery by carrying cell-destroying chemicals specifically to cancer cells.

Specific Aim: To isolate and amplify an RNA aptamer that binds specifically to FGF8b.

Budget/Ordering: Shawn Piasecki of the Keatinge-Clay Lab provided last year’s his-tagged

FGF8b stock and will continue to do so. Thus, pricing information from this source in particular is

unknown, but untagged human FGF8b may be purchased in quantities of 100 ug for $187.16 from US

Biological.

Figure 1. This crystallographic image depicts an aptamer (in green) binding

to a target molecule, taken from Limson (2013).

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Figure 1. Diagram of the SELEX selection

process. A large pool of random RNA is

incubated with a target, and binding species

are isolated. These species are amplified and

used to start another cycle, taken from Cass

(2011)

Introduction:

Few diseases today can match the prevalence and lethality of cancer. As their populations

age and become more sedentary, even developed countries like the United States have to deal with rising

cancer rates. Recent estimates suggest that as many as 7.6 million people died of cancer in 2008, making

it the leading cause of death for that year (Jemal et al, 2011). Prostate cancer in particular has been

identified as the second most common form of cancer in men, and it was responsible for 28,088 deaths in

2009 (US Cancer Statistics Working Group, 2013). Although the word “cancer” actually refers to a

family of related diseases rather than a specific illness, all cancers involve unregulated cell division.

Although a series of biochemical checkpoints normally ensures that a “malfunctioning” cell undergoes

programmed cell death (called apoptosis), cancerous cells have genetic mutations that allow them to

bypass these measures (Croce, 2008).

Studies have indicated that a family of proteins, known as Fibroblast Growth Factors, is directly

related to the development of cancers. Fibroblast growth factors in vertebrates can range from 17 to 34

kilodaltons (kDa) in size and play an important role in the development of embryos: each FGF is

responsible for a different aspect of growth. Mammalian growth factor (FGF8b), the growth factor that

was targeted in this study, has a weight of 24,148 Da and affects central nervous system development and

limb development, as well as gastrulation. . FGF8b is found as a monomer. There are 22 members in the

human FGF family, and all require heparan sulfate in order to bind to FGF receptors. Most FGFs

(including FGF8b) are excreted by cells and are typically found in the extracellular matrix (Ornitz and

Itoh, 2001).

A method of successfully detecting

overexpressed FGFs in cells could help oncologists

locate early-stage cancer patients before treatment

becomes difficult. Increased expression of FGF8b

in particular is a known indicator of prostate cancer

(Kwabi-Addo, 2004). Despite attempts by

numerous labs specializing in

immunohistochemistry, attempts to create custom

antibodies to locate this target have failed due to

low specificity and high costs. For this reason, an

aptamer with a high binding affinity for an FGF

would be highly desirable.

Aptamers are oligeonucleotides (or

sometimes peptides) that bind to a specific

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molecule. Aptamers are superior to antibodies for a number of reasons, most of which stem from the way

they are created. Aptamers are developed through an in vitro selection process involving the isolation of

high-binding species from a large pool in a process known as Systematic Evolution of Ligands by

Exponential enrichment), or SELEX (Ni, 2011). In a standard SELEX selection round, binding species of

oligeonucleotides are incubated with a target, filtered out, and reamplified for another round as is shown

in figure 1(adapted from Proske, 2005) . In vitro selection (meaning in artificial conditions AKA test

tubes) means that aptamers can be developed for a wider range of conditions than antibodies (which have

to be made in vivo, or in the body). Aptamers are also significantly cheaper and easier to make, and they

have a longer shelf life. Furthermore, aptamers cannot provoke unwanted immune responses, and their

smaller size makes them more specific by making them better transduction groups for biosensors (Keefe,

2010). Figure 2 shows how aptamers and antibodies can be combined to form extremely potent and

highly specific therapeutic agents that have greater pharmacokinetic properties than their parents

(Wuellner, 2010).

Dr. Anne Moon at Geisinger Health System in Danville, PA has specifically requested an aptamer

for FGF8b to be used in localization studies. There are many other labs working with FGF8b, such as the

Roy-Burman Lab at USC (Chen, 2005). The Presta Lab at the University of Brescia in Italy has studied

the soluble pattern recognition receptor long petraxin-3 as an antagonist of FGF8b, acting to prevent it

from binding to its receptors and inhibiting the development of tumor cells as a result (Leali, 2011).

Before beginning SELEX, it is important to confirm that aptamers have not already been made

for the desired target. As of September 2013, no aptamers had been isolated for FGF8b. It is also critical

to consider the downstream applications of the aptamer when selecting, such as the location where the

aptamer will be used. In this case the desired aptamer, if found, would be used in the human body as a

diagnostic for cancer. Aptamers could be fluorescently tagged prior to injection into the body, so that they

can be used to locate small tumors. FGF8b, while not known specifically for binding nucleic acids, has a

positive charge in solution, and its isoelectric point is greater than 10. Oligeonucleotides like RNA have a

slight negative charge, so it was assumed that RNA species would bind easily to the target even though

FGF8b does not typically bind to nucleic acids in nature.

There are different selection methods that can be used to isolate high-binding RNA species: these

include magnetic bead, columns, and filters. This lab uses bead-based selection which involves

immobilizing a target protein on a set of beads in a tube. In order for this to work, the target protein must

first be “tagged” with a special chemical group such as histadine. Histadine has a high affinity for nickel

and will readily stick to nickel-coated beads.

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Once an aptamer has been found, a self-assembling aptamer-micelle nanostructure can be made to

internalize the aptamer into the cell (Wu, 2010). This aptamer could then be injected into the body to

detect prostate cancer.

Alternatively, this

aptamer could be

attached to a

nanoparticle to

generate a targeted

nanoparticle. A

chemotherapeutic

agent such as

docetaxel could then be encapsulated within this structure to increase cytotoxicity (Xiao et al, 2012).

Materials and Methods:

The nickel-nitrilotriacetic acid (Ni-NTA) beads were stored in a buffer containing 200 mM NaCl,

1 mM MgCl2, and 10 mM tris(hydroxymethyl)aminomethane (tris) at a pH of 7.4 at a temperature of 4°C.

Table 1 shows the initial conditions for each round (table 2 gives the RNA : target ratio and wash

volume/numbers). Prior to the actual selection, the beads that were used were pre-washed three times with

the selection buffer. The mFGF8b was then allowed to immobilize on the beads for 30 minutes at room

temperature. While this occurred, the previous round’s N40B RNA pools (R0 N40B pool for first round)

were incubated at 65°C for 3 minutes with selection buffer and allowed to cool (so that they could fold

into their most stable structure). During the rounds with negative selections, these pools were first

incubated with a tube of beads containing no proteins. The buffer from this process was then drained out

and added to the beads with the target (which removed the RNA that was sticking to the beads rather than

the target). The beads with the protein were then rewashed with selection buffer three times (to remove

the unbound protein) and set to incubate with the RNA pool. The plus/minus selection conducted in round

4 followed a similar process to the negative selection except that the negative RNA (with only beads) was

also washed off, reverse transcribed, and amplified by cycle course. This was compared with a normal

cycle course from the positive (regular) selection in order to confirm that the RNA was primarily binding

to the target rather than the beads. Afterwards, the unbound pool was isolated from the beads. These

beads were then washed with selection buffer, and the solution was then removed and labeled as W1

(wash one). This was repeated until the last wash. Here, the beads along with the buffer were placed in a

new tube before the wash was removed (to get rid of plastic binding species). The RNA species that were

Figure 2. Aptamers and Antibodies working together. Aptamers can be

linked to antibodies for greater accuracy and survivability, adapted from Wuellner

(2010).

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actually bound to the beads were then removed by vortexing in hot (80°C) water. The unbound pool

(WO), last wash (W3 or W5), and the eluted binding pool (E1) that resulted from these selections were

concentrated with standard ethanol precipitation using 1/10th volume 0.3 M NaOAc at pH 5.2, 3 uL

glycogen, and 2.5 volumes pure ethanol. This was chilled at -80°C for fifteen minutes and centrifuged at

max speed for ten minutes. The supernatant was removed, and 400 uL of chilled 70% ethanol was added

to the precipitate which was chilled on ice for two minutes, re-centrifuged for five minutes, and dried in a

speedvac for ten minutes. All subsequent precipitations were performed in the same way. The pellet was

resuspended in 20 uL diH2O (at a high enough concentration to be reverse transcribed).

A 20 uL mixture containing concentrated RNA (from W0, W3/W5, and E1), 20 uM N40B

reverse (R) primer, and 0.5mM deoxyribonucleotides (dNTPs) was heated at 65°C for 5 minutes and

cooled to room temperature. A mixture of this and First Strand buffer, 0.01M DTT, and 1 uL SuperScript

II Reverse Transcriptase (SSII RT) was then incubated at 42°C for fifty minutes, inactivated at 70°C for

fifteen minutes, and cooled to 4°C for storage (reverse transcription, RT). A 100 uL cycle course

polymerase chain reaction (PCR) was performed to determine the optimal number of cycles for large

scale (ls) PCR: a mixture of PCR buffer (10 mM Tris-HCl pH 8.3, 50 mM KCl, 2 mM MgCl2), 0.2 mM

dNTP, 0.4 uM 88.35 forward (F) primer, 0.4 uM 20.35 reverse (R) primer, 2 uL single stranded (ss) DNA

from RT (three different samples), 4 U Taq DNA polymerase, and diH2O was put through twenty cycles

of PCR. The conditions were: 94°C for two minutes (only once), 92°C for forty-five seconds, 54°C for

forty-five seconds, 72°C for one minute, repeat nineteen times, chill at 4°C. A 5 uL sample was removed

from each of the three at six cycles, nine cycles, twelve cycles, fifteen cycles, and twenty cycles and

mixed stained with 1 uL ethidium bromide (EtBr). These samples were then run on a 3.8% agarose gel for

thirty-five minutes at 120 volts along with a no template control (NTC) and a DNase control (only in

round 2, using “purified” RNA from the last round). The number of cycles that produced the best band for

E1 was chosen for large scale PCR.

The lsPCR contained the same reagents as the cycle course but divided into six tubes.

These were cycled based on the results of the cycle course and then run on a gel (along with another

NTC). The lsPCR was concentrated with ethanol precipitation and resuspended in 20 uL of diH2O. This

precipitate was used as a template in a 20 uL transcription (TNX) reaction along with other reagents:

TNX buffer, 10 mM DTT, 7.5 uM of each nucleoside triphosphate (ATP, CTP, UTP, and GTP in that

order), ~800 ng double stranded (ds) DNA from lsPCR, diH2O, and 2 uL T7 RNA polymerase enzyme

solution. This was incubated at 37°C overnight and combined with 1 uL of DNase I enzyme solution for

another fifteen minutes at 37°C. The enzyme was quenched with 21 uL (one reaction volume) of blue

denaturing dye containing 7 M urea, EDTA, and bromophenol blue. This was denatured by heating for

three minutes at 65°C and added to a polyacrylamide gel made with 8% denaturing acrylamide, 25 uL of

Dugger FGF8-b

Table 1. This table shows the selection

conditions used in each round.

tetramethylethylenediamine (TEMED), and 0.04% ammonium persulfate (APS). The gel was run at 450

volts for one hour, and the transcription product was located with a UV light and TLC plate. This product

was cut out and placed in 850 uL of TE for overnight elution. In R2, the gel was instead crushed and

mixed with 400 uL of 0.3M NaOAc and TE. This was heated at 80°C for five minutes and centrifuged at

max speed for one minute. The eluate was removed and added to a red dot Ultrafree-MC spin filter tube

for three minutes of centrifugation filtration. This was repeated a second time with the gel chunks to skip

the overnight elution step and achieve a greater yield. After elution, ethanol precipitation was performed

as before and the pellet was resuspended in 30 uL of diH2O. This precipitated RNA was then quantitated

with a Nanodrop spectrophotometer and used for the next round (four rounds were performed as of this

report with one in progress).

Progress, Results, and Discussion:

The selection process as a whole involved repeating several rounds to isolate and amplify highly

specific RNA before performing a binding assay and sequencing. Early on, there were very few points

where it was possible to check for error. The ethanol precipitations of the round one E1, W0, and W3 all

produced pellets, indicating the possible presence of RNA (although it could have been only salt). In

contrast, the same step in the second round produced only one pellet which was found in W0. However,

missing pellets do not mean absent RNA (they are usually just too small to see), and subsequent cycle

course PCRs in both rounds provided decent results.

In all cycle courses, the W0 showed the earliest amplification and the final wash showed the

latest. This was expected because the majority of the RNA did not bind to the target. Instead, this RNA

R1 – R3 R4 – R5

Pool N71 (dsDNA = 132

bp, RNA = 112 nt)

N71

Incubation Time/

Temperature

25 minutes at 37°C 25 minutes at 37°C

Buffer and pH 1X PBS pH 7.4 1X PBS pH 7.4

Salt and Ion

Concentration

1 mM Phosphate,

13.7 mM NaCl, 2.7

mM KCl, 5 mM

MgCl2

1 mM Phosphate,

13.7 mM NaCl, 2.7

mM KCl

Dugger FGF8-b

ended was washed away in W0 and amplified the most out of all the washes. By the last wash, most of the

RNA was washed off with the remaining material staying attached to the target. That is why the elution

showed slightly more amplification than the last wash. The R1 cycle course had no NTC bands, but the

R2 cycle course had a very faint line that was similar to its cycle 9 E1. Rounds 3 and 4 produced no NTC

bands, but round 5 had a thin one. Also, the DNase control showed up in every round it was performed

(R2, R3 and R5) which meant that some residual template DNA from the prior round was not completely

eliminated, presumably due to the low activity of the DNase I enzyme. These issues were judged minor

enough to continue with the selection, so an lsPCR was performed with both (14 cycles for R1 and 12

cycles for R2). The picture of the R1 lsPCR was lost and is not included here, but it showed a bright E1

band with no overamplification and no NTC band. The R2 lsPCR turned up with the same results. In both

cases, this meant that the selection could be continued without any change in selection conditions. In both

of the new rounds (see problems encountered for explanation), a new pool (N71) was used. Both times,

double bands were produced during over amplification. In order to ensure that this problem would not

arise in the lsPCR (which would have entailed starting over), a very small number of cycles was used in

both new lsPCRs (9 cycles both times). Fortunately, both lsPCRs showed single and clear bands. In the

third round, the cycle course was too faint to determine cycles for lsPCR (see Problems Encountered).

Therefore, the selection was started over for round 3 (performing more cycles was impossible because the

thermocycled tubes had already been thrown away and the reverse transcription product was not found) .

The new cycle course was odd in that it produced no bands in cycles 6 and 9 but created obvious smears

from cycle 12 onward. For this reason, 10 cycles were used for the R3 lsPCR to be conservative.

In each round, the lsPCR product was precipitated, transcribed, and then run on a PAGE gel at

450V. The R3 lsPCR still turned up smeared, so the experiment was stopped here due to time contraints.

The previous rounds’ bands were visualized in UV light. Any “shadows” that were produced indicated the

presence of RNA: the UV rays that caused the TLC plate to fluoresce were absorbed by the RNA. The R1

PAGE gel produced a very clear shadow concentrated in one location. This made it easy to cut out the

RNA gel chunks for elution. The R2 band looked unusual and had a warped, asymmetrical appearance.

Despite this, it was still clear enough to be demarcated and cut out in multiple pieces.

After this process, the eluted gels were precipitated with ethanol and resuspended in 30 uL of

diH2O. A nanodrop spectrophotometer was then used to measure the concentration of RNA in the samples

by measuring absorbance. The R1 RNA had a yield of 2605.20 pmol while the R2 RNA had a yield of

2607.19 pmol but these results were useless (see Problems Encountered). Thus, the selection conditions

for these rounds are not included in the table. The new R1 RNA had a yield of 1676.69 pmol, and the new

R2 RNA pool had a yield of 2048.29 pmol. In the fall, a third round was successfully conducted that

produced 1290.82 pmol of RNA. This round amplified very quickly in the cycle course but still provided

NTC

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good bands (not overamplified or too thin) in the large scale. In the fourth round, a plus/minus selection

was conducted in order to give a rough estimate of how well the RNA was binding to the target (as

opposed to the beads). The positive cycle course elution was much brighter than the negative cycle course

elution (which only produced bands in the 20th cycle in the elution, see figures 16 and 17). This proved

that most of the RNA was sticking to the FGF8b rather than the Talon beads (the negative elution would

be much brighter in this case). Afterwards, the large scale PCR produced a clear band of the appropriate

length with very faint primer-dimers. The primer-dimers were not determined to be an issue; however,

because the main bands did not overamplify the primer-dimers could be removed during the PAGE gel

purification process (the shorter bands travel much further than the longer bands and are not excised). The

Round 4 transcription was extremely effective (probably due to using the 42°C 2 hour reaction rather than

the 37°C overnight reaction), with a concentration of 97.8 pmol/uL. Since the Nanodrop cannot

accurately measure concentrations over 71.3 pmol/uL, the concentration was diluted to 24.7 pmol/uL with

20 uL of water. In round 5, some of the E1 appeared to leak out of the tube prior to the first ethanol

precipitation (after binding and selection). This was probably part of the reason that no bands were

observed in the cycle course until the 20th cycle (this band was overamplified). The lsPCR for this round

failed to produce any bands at all, so another lsPCR was run for 20 cycles using the remaining reverse

transcription product. The results of this are seen in figure 21. Multiple cycle course/large scale PCRs

were necessary to correct the issues that arose after this step (see figures 19-23 and Problems Encountered

for a full explanation). Eventually, round 5 produced 1816 pmol of RNA.

All lab figures are on following 4

pages

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Figure 3. R1 Cycle Course. This 3.8% agarose

gel was run at 100V for 40 minutes. The bands

were the correct length (96 bp). Cycle 20 showed

unusual amplification (the primers started laying

down incorrectly), but the NTC was clear.

Fourteen cycles were used for the large scale

(cycle 12 was too faint and cycle 15 over-

amplified).

Figure 4. R2 Cycle Course. The bands were the correct length

(still 96 bp). A very faint band was visible in the NTC (in blue

box), but this was not enough to warrant starting over (cause

was determined to be pipetting error, not contamination) so the

process was continued (12 cycles chosen for lsPCR). The DNase

control showed excessive amplification, meaning that the DNase

I enzyme removed very little template DNA.

Figure 5. R2 lsPCR. The

round 2 lsPCR produced a

good band (96 bp) at 12

cycles with no smearing

and did not have an NTC

band like the previous

cycle course.

Figure 6. New R1 Cycle Course. Cycle 12

seemed to produce double bands in the E1,

but the single bands in the earlier cycles

were the correct length (112 bp). 9 cycles

were chosen for large scale.

Figure 7. New R1

lsPCR. This lsPCR

produced no double

bands and was the

correct length (132 bp).

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Figure 12. R3 lsPCR. Although

difficult to see, the blue box

indicates a very faint smear. This

was very unusual and showed that

the sample was probably

contaminated (not

overamplification).

Figure 9. New R2 lsPCR.

This lsPCR (9 cycles) also

showed no double bands. A

DNase control was performed

and indicated that the DNase

was not active enough to

destroy the DNA template

from the previous round.

Figure 8. New R2 Cycle Course. This

ccPCR also showed double bands and did not

show up very well under UV light (likely due

to the thickness of the gel). Cycles chosen = 9

***NOTE***: Figures 10-11

were lost in this file but are

available on progress report #1

(textboxes and figures are on this

page but are invisible/irretrievable

for some reason).

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Figure 15. Illustration of PAGE gel. This

crude picture shows what an 8% acrylamide

PAGE gel should look like under UV light.

The boxes at the top are the wells and the

crescent is the blue dye. The dark circle is

the shadow created by the RNA above the

TLC plate. This N71 RNA is 112 nt long.

Figure 13. R3 ccPCR Fall. This gel

amplified very early (cycle 9 was

already very bright), so only 6

cycles were used for the large scale

PCR.

Figure 14. R3 lsPCR Fall. This gel

produced a good band (not too bright) and

no NTC band at 6 cycles.

Figure 16. R4 + ccPCR Fall. This gel

amplified just as quickly as R3, and

produced only one single band. Cycles

chosen = 6.

Figure 17. R4 - ccPCR Fall. This

gel only had bands in cycle 20 in

the last wash and elution. This was

a good sign that the RNA was not

binding to the beads.

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Figure 18. R4 lsPCR Fall. The lsPCR

produced a primer-dimer at 6 cycles but

was otherwise good.

Figure 19. R5 ccPCR Fall. This gel took a very long time to

produce a solid band. 18 cycles were chosen since the 20th

cycle was overamplified.

Figure 20. R5 failed lsPCR Fall. This

lsPCR failed to produce any bands. It is

likely that not enough cycles were

performed to amplify anything. (18

cycles).

Figure 21. R5 lsPCR-2 Fall. A second

lsPCR was performed with the same

reagents (including RT product) using 20

cycles. This yielded no product

amplification, providing only primer-

dimers (60-70 nt long).

Figure 22. R5 ccPCR-2 Fall. Another

RT was performed with the original R5

elution. A positive control (W0 from R2)

was used to confirm that there was no

issue with reagents (nonfunctional Taq).

14 cycles were chosen.

Figure 23. R5 lsPCR-3 Fall. This lsPCR (14 cycles)

appeared warped. The gel used to run the DNA was

not heated enough, so the lsPCR band appears too

long even though it is actually the correct length.

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Problems Encountered:

Near the end of the second round, a major issue was found that demanded the restart of the entire

selection: The targets that had been used for both rounds were not his-tagged. This meant that the proteins

were immediately washed off the beads after the target immobilization step, and that the RNA species

that were isolated were actually bead-binding species. Even when a negative selection was performed in

round 2, the concentration of RNA that was isolated at the end remained relatively high. This meant that

the negative selection was ineffective and that there was no way to continue on to the next round. Had

this problem not been found, then any aptamer that might have been discovered would have been useless:

this species would have a high affinity for nickel beads rather than the actual target, FGF8b.

The reason for this serious error was very simple: the tubes were mislabeled. The box containing

the tubes contained a note identifying the samples as his-tagged FGF8b. The note also described the

volume of the samples and the amount of target present in each tube. This all seemed to indicate that the

tubes inside the box were the correct ones. However, two major clues demonstrated that this was not the

case: the tubes were colored and the volume did not match the description. This problem can be avoided

in the future by more careful labeling as well as by confirming the identity of the sample with the primary

investigator or with a peer. The gels were somewhat difficult to see in the new rounds, but this was likely

due to the amount of gel that was used (too thick) which can be easily fixed in the future by pouring gels

of optimum thickness. The failed R3 ccPCR may have resulted from an error in the ethanol precipitation

prior to reverse transcription (pellets were nearly invisible). The ladder still turned up but all samples

(including the W0 samples) were practically invisible, so the error had to have been with the thermocycler

machine (unlikely), the ethanol precipitation, or one of the reagents. To correct for this in the second

attempt, the 70% ethanol washing step in the precipitation process removed (it may have washed away

RNA). Both the cycle course and the lsPCR produced smears with no clear bands, so the sample may

have been. As a result, the round was started over from selection using fresh aliquots of reagents. This

round (which was performed in the fall semester) produced very clear gels with no major issues. The

bands in the ccPCR amplified almost immediately (already very bright in cycle 9), so the next round had

much more stringent conditions.

In R5, numerous issues were encountered. The elution from the first cycle course did not amplify

until the 20th cycle, and a very faint band appeared in the NTC column (figure 19). The NTC band was

due to poor pipetting technique rather than contaminated aliquots (confirmed in the lsPCR NTC). Only 18

cycles were used in the lsPCR, and ethanol precipitation was conducted at the same time that the gel was

run. When the gel showed no amplification for the lsPCR (figure 20), it was clear that not enough cycles

had been used. However, it was impossible put the DNA through more cycles as it had already been

precipitated. Thus, a second lsPCR was conducted using the same RT product with 20 cycles. However,

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only primer dimers appeared (figure 21). This indicated that there was very little DNA in the RT product

to begin with (what little was present was likely used in the initial ccPCR). Therefore, a second RT was

performed with the R5 elution to generate a higher yield of RNA. This product was used to run a second

cycle course (along with a positive control to ensure that the Taq enzyme was still functioning). This

cycle course amplified much earlier (figure 22) and produced no NTC bands. Thus, the new RT product

was used in a third lsPCR. This band appeared longer than expected, but this was due to improperly

heated gel that resulted in distorted bands (figure 23). It was this product that was finally transcribed and

quantitated.

Conclusion and Future Work:

The overarching purpose of this selection is to find a species of RNA with a high binding affinity

for mammalian fibroblast growth factor 8 b isoform. During the first two rounds, high concentrations of

RNA with this property were supposedly isolated and quantified. However, this was not the case due to

the lack of his-tagged targets, so the selection process was restarted and will be continued for

approximately six rounds. The original plan of completing six rounds by the end of April was no longer

tangible due to the problems encountered with the first two rounds, so the plan has now changed to

finishing the selection at the beginning of the fall semester. With each subsequent round, a greater number

of washes/ greater total wash volume will be used to increase stringency. In addition to this, the protein to

target ratio was changed to 400 pmol RNA: 200 pmol target after the first round and will be lowered

further every two or three rounds depending on the results. Furthermore, negative selections will be

performed for every future round to avoid isolating bead-binding species. The incubation time and

temperature will not be changed at all unless the yields get too small, and the selection buffer will not be

altered in any way. Afterwards, a binding assay will be performed to determine what the

oligeonucleotides are actually binding to. Hopefully, subsequent rounds will show more binding to

FGF8b than to the beads. Upon completion of this, the RNA species will be sequenced to find any motifs

that cause them to bind so that a more effective structure can be developed. Ultimately, an aptamer may

be identified that can help with the identification and treatment of prostate cancer.

Round Pool : Target

(pmol : pmol)

Washes

(# x Volume)

No. of PCR cycles necessary to

amplify selected pool

Amount of recovered pool

(pmol)

1 400 : 400 5 x 500 uL 9 980

2* 400 : 200 4 x 1000 uL 9 1197

3* 400 : 200 5 x 1000 uL 6 754

4** 400 : 100 10 x 1000 uL 6 1139

5* 400 : 100 10 x 1000 uL 14 1816

Table 2: Summary of anti-FGF8b aptamer

selection rounds. * = Negative selection

** = Negative selection and +/- selection

Dugger FGF8-b

References:

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