Another Realization of Aqueous Computing with Peptide Nucleic Acid

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 • 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 - handles an aqueous sol’n of general-purpose memory molecules with a small set of elementary laboratory operations - fits to solve a certain pattern NP-complete problem - copy a memory state upon a DNA seq by whiplash PCR

3. Aqueous Algorithm • Pour(n): divide the solution into n tubes. • Unite: mix n tubes into one. → resulting sol’n • SetToZero(k) : set the kth bit of all memory molecules in that tube to be 0. • MaxCountOfOnes : find the max number of 1’s in one memory molecule from that tube.

4. Aqueous memory

5. Structure of PNA and DNA

6. 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 Thermal stability - 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 Long lasting - resistant to enzymatic degradation  because their hybrid chemical structure is not recognize nucleases or proteases. - Stable over a wide pH range

8. Characteristics of PNA Binding 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 * Hybridization of PNA can also occur in the absence of Mg2+, 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 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

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

11. Strand Displacement

12. Memory State Copy

13. 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 - Need a control experiment

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