Another Realization of Aqueous Another Realization of Aqueous Computing with Computing with

Peptide Nucleic AcidPeptide Nucleic Acid

August 8, 2001 August 8, 2001

Park, Ji-YoonPark, Ji-Yoon

Masayuki Yamamura, Yusuke Hiroto, and Taku MatobaMasayuki Yamamura, Yusuke Hiroto, and Taku Matoba

AbstractAbstract

Elementary operation for aqueous computing with PNA and realizElementary operation for aqueous computing with PNA and realize one bit memory for a feasibility study to confirm strand displacee one bit memory for a feasibility study to confirm strand displacement by PNA ment by PNA

Aqueous computingAqueous computing

- code design free molecular computing- code design free molecular computing

- handles an aqueous sol’n of general-purpose memory molecules - handles an aqueous sol’n of general-purpose memory molecules

with a small set of elementary laboratory operationswith a small set of elementary laboratory operations

- fits to solve a certain pattern NP-complete problem- fits to solve a certain pattern NP-complete problem

- copy a memory state upon a DNA seq by whiplash PCR - copy a memory state upon a DNA seq by whiplash PCR

Aqueous AlgorithmAqueous Algorithm

Pour(n): divide the solution into Pour(n): divide the solution into

nn tubes tubes. Unite: mix Unite: mix nn tubes into one. tubes into one.

→ → resulting sol’n resulting sol’n SetToZero(SetToZero(kk))

: set the : set the kkth bit of all memory th bit of all memory

molecules in that tube to be 0.molecules in that tube to be 0. MaxCountOfOnesMaxCountOfOnes

: find the max number of 1’s in : find the max number of 1’s in

one memory molecule from one memory molecule from

that tube. that tube.

Aqueous memoryAqueous memory

Structure of PNA and DNAStructure of PNA and DNA

Peptide Nucleic acid(PNA)Peptide Nucleic acid(PNA)

Analogs of DNA in which the phosphate backbone is replaced with an Analogs of DNA in which the phosphate backbone is replaced with an uncuncharged “peptide-like” backboneharged “peptide-like” backbone

The achiral backbone is made of The achiral backbone is made of repeating N-(2-aminoethyl)-glycine units repeating N-(2-aminoethyl)-glycine units linked by amide bondslinked by amide bonds

No deoxyribose or phosphate groupsNo deoxyribose or phosphate groups are present are present

Backbone is unchargedBackbone is uncharged

PNA to hybridize to complementary RNA or DNA with higher specificity PNA to hybridize to complementary RNA or DNA with higher specificity and affinity, making PNA and affinity, making PNA good candidates for the inhibition of gene expregood candidates for the inhibition of gene expressionssion

Characteristics of PNACharacteristics of PNA

Thermal stabilityThermal stability

- The lack of charge repulsion between the PNA strand and the DNA or RNA - The lack of charge repulsion between the PNA strand and the DNA or RNA stronge stronger binding between PNA/DNA or PNA/RNAr binding between PNA/DNA or PNA/RNA

- A higher thermal stability of the duplexes- A higher thermal stability of the duplexes

Long lastingLong lasting

- resistant to enzymatic degradation- resistant to enzymatic degradation

because their hybrid chemical structure is not recognize nucleases or proteases. because their hybrid chemical structure is not recognize nucleases or proteases.

- Stable over a wide pH range- Stable over a wide pH range

Characteristics of PNACharacteristics of PNA

Binding independent of salt concentrationBinding independent of salt concentration

** The Tm of PNA/DNA duplexes is independent of salt concentration The Tm of PNA/DNA duplexes is independent of salt concentration

* At a low ionic strength PNA can be hybridized to a target sequence at temperatures * At a low ionic strength PNA can be hybridized to a target sequence at temperatures at which normal DNA hybridization is inhibitedat which normal DNA hybridization is inhibited

* Hybridization of PNA can also occur in the absence of Mg* Hybridization of PNA can also occur in the absence of Mg2+2+, a factor that further in, a factor that further inhibits DNA/DNA duplex formation.hibits DNA/DNA duplex formation.

Triplex formationTriplex formation - PNA oligomers containing only thymines(T) and cytosines(C) often prefer to bind a 2- PNA oligomers containing only thymines(T) and cytosines(C) often prefer to bind a 2

PNA/1DNA stoichiometry resulting in high stability PNA/1DNA stoichiometry resulting in high stability

Strand displacementStrand displacement - - Homopyrimidine PNA oligomers displace a DNA strand from DNA/DNA duplex to foHomopyrimidine PNA oligomers displace a DNA strand from DNA/DNA duplex to fo

rm a local PNA/DNA/PNA triplex and a D-looprm a local PNA/DNA/PNA triplex and a D-loop

Material and MethodsMaterial and Methods

1. For 2 pmol DNA, we added (1) 0.0 pmol, (2) 100 pmol, (3) 200 pmol PNA1. For 2 pmol DNA, we added (1) 0.0 pmol, (2) 100 pmol, (3) 200 pmol PNA

2. Incubate in 17 ul 1 2. Incubate in 17 ul 1 × TE buffer at 37ºC for 1 hr × TE buffer at 37ºC for 1 hr

3. Add 15 units of Xba I for all three samples 3. Add 15 units of Xba I for all three samples

4. Incubate in 20 ul 1 4. Incubate in 20 ul 1 × M buffer at 37ºC for 30 min× M buffer at 37ºC for 30 min

5. Run samples in a 10% polyacrylamide gel electrophoresis 5. Run samples in a 10% polyacrylamide gel electrophoresis

PNA(15 bp): N-CCGCPNA(15 bp): N-CCGCTCTAGATCTAGAACTAG-C ACTAG-C DNA(2 pmol)DNA(2 pmol) Xba I(Takara)Xba I(Takara)

ResultResult

M: 20 bp ladder(Takara)M: 20 bp ladder(Takara)

NC: Negative controlNC: Negative control

Lane 1: DNA(2 pmol)Lane 1: DNA(2 pmol)

Lane 2: DNA(2 pmol) + PNA(100 pmol)Lane 2: DNA(2 pmol) + PNA(100 pmol)

Lane 3: DNA(2 pmol) + PNA(200 pmol) Lane 3: DNA(2 pmol) + PNA(200 pmol)

MM 11NCNC 22 33

10% Polyacrylamide gel electrophoresis10% Polyacrylamide gel electrophoresis

200bp200bp

100bp100bp

140bp140bp

240bp240bp

100

pmol

100

pmol

200

pmol

200

pmol

0 pm

ol

0 pm

ol

Strand DisplacementStrand Displacement

Memory State CopyMemory State Copy

DiscussionDiscussion

The molecular weight of displaced DNA is increased by 15 bp PNThe molecular weight of displaced DNA is increased by 15 bp PNAA

We could not find the optimum condition for strand displacementWe could not find the optimum condition for strand displacement

- Purity of both DNA and PNA - Purity of both DNA and PNA

- Need a control experiment- Need a control experiment

ConclusionConclusion

Aqueous computingAqueous computing Biomolecular realization with PNA-DNA hybrid Biomolecular realization with PNA-DNA hybrid Preliminary experiment with one bit memory Preliminary experiment with one bit memory An idea to copy memory state An idea to copy memory state