1. Another Realization of Aqueous Computing with Peptide Nucleic Acid August 8, 2001 Park, Ji-Yoon Masayuki Yamamura, Yusuke Hiroto, and Taku Matoba

2. Abstract Abstract  Elementary operation for aqueous computing with PNA and realize one bit memory for a feasibility study to confirm strand displacement by PNA  Aqueous 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 operations with 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

3. Aqueous Algorithm  Pour(n): divide the solution into n tubes n tubes.  Unite: mix n tubes into one. → resulting sol’n → resulting sol’n  SetToZero(k) : set the kth bit of all memory : set the kth bit of all memory molecules in that tube to be 0. molecules in that tube to be 0.  MaxCountOfOnes : 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.

4. Aqueous memory

5. Structure of PNA and DNA Structure of PNA and DNA

6. Peptide Nucleic acid(PNA) Peptide Nucleic acid(PNA)  Analogs of DNA in which the phosphate backbone is replaced with an uncharged “peptide-like” backbone  The achiral backbone is made of repeating N-(2-aminoethyl)-glycine units linked by amide bonds  No deoxyribose or phosphate groups are present  Backbone is uncharged  PNA to hybridize to complementary RNA or DNA with higher specificity and affinity, making PNA good candidates for the inhibition of gene expression

7. Characteristics of PNA Characteristics of PNA Thermal stability - The lack of charge repulsion between the PNA strand and the DNA or RNA  stronger binding between PNA/DNA or PNA/RNA - The lack of charge repulsion between the PNA strand and the DNA or RNA  stronger binding between PNA/DNA or PNA/RNA - A higher thermal stability of the duplexes - A higher thermal stability of the duplexes Long 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

8. Characteristics of PNA Characteristics of PNA Binding 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 which normal DNA hybridization is inhibited * At a low ionic strength PNA can be hybridized to a target sequence at temperatures at which normal DNA hybridization is inhibited * Hybridization of PNA can also occur in the absence of Mg 2+, a factor that further inhibits DNA/DNA duplex formation. * Hybridization of PNA can also occur in the absence of Mg 2+, a factor that further inhibits DNA/DNA duplex formation. Triplex formation - PNA oligomers containing only thymines(T) and cytosines(C) often prefer to bind a 2PNA/1DNA stoichiometry resulting in high stability - PNA oligomers containing only thymines(T) and cytosines(C) often prefer to bind a 2PNA/1DNA stoichiometry resulting in high stability Strand displacement - Homopyrimidine PNA oligomers displace a DNA strand from DNA/DNA duplex to form a local PNA/DNA/PNA triplex and a D-loop - Homopyrimidine PNA oligomers displace a DNA strand from DNA/DNA duplex to form a local PNA/DNA/PNA triplex and a D-loop

9. Material and Methods 1. For 2 pmol DNA, we added (1) 0.0 pmol, (2) 100 pmol, (3) 200 pmol PNA 2. Incubate in 17 ul 1 × TE buffer at 37ºC for 1 hr 3. Add 15 units of Xba I for all three samples 4. Incubate in 20 ul 1 × M buffer at 37ºC for 30 min 5. Run samples in a 10% polyacrylamide gel electrophoresis  PNA(15 bp): N-CCGCTCTAGAACTAG-C  DNA(2 pmol)  Xba I(Takara)

10. Result Result M: 20 bp ladder(Takara) M: 20 bp ladder(Takara) NC: Negative control NC: 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) M 1 NC 2 3 10% Polyacrylamide gel electrophoresis 200bp 100bp 140bp 240bp 100 pmol 200 pmol 0 pmol

11. Strand Displacement Strand Displacement

12. Memory State Copy Memory State Copy

13. Discussion Discussion  The molecular weight of displaced DNA is increased by 15 bp PNA  We 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

14. Conclusion Conclusion  Aqueous computing  Biomolecular realization with PNA-DNA hybrid  Preliminary experiment with one bit memory  An idea to copy memory state