1. AQUEOUS COMPUTING - Writing on Molecules - T. Head, M. Yamamura, and S. Gal Binghamton University

2. 7/9/99CEC'992 1. Introduction u The only way to compute with DNA? 1 design sequences for DNA molecules 2 order many custom DNA molecules 3 anneal and filter ( 4 if failure goto 1 ) ↓ u Aqueous computing –framework for using molecular memory –laboratory implementation

3. 7/9/99CEC'993 Molecular Memory MemoryLSIHD Address wired gridhead pos. Content electronicmagnetic 1. molded together 2. fixed on solid materials 3. serial processing AQUEOUS DNA specific subsequence markings on molecules 1. individual access 2. randomize location 3. parallel processing easily separate mix again

4. 7/9/99CEC'994 2. Mathematical Basis u Common algorithmic problem (CAP) –a description of the pattern of the problem u Aqueous algorithm –a way to use molecular memory

5. 7/9/99CEC'995 Common algorithmic problem u CAP given S: finite set F ⊂ 2 S (the forbidden subsets) find the largest cardinal number n for which there is a subset T of S for which: |T|=n, ∀ U ∈ F U ⊂ T. –NP-complete problems having the CAP pattern »maximum independent set »minimum vertex cover »Hamiltonian cycles »Boolean satisfiability, etc.

6. 7/9/99CEC'996 Example u Maximum independent set problem given: G=(V, A) (the arcs are forbidden) find max |T| s.t. T ⊂ V, ∀ x,y ∈ T, {x,y} ∈ A Find max # of animals you can keep in one cage?

7. 7/9/99CEC'997 Aqueous Algorithm Initialize; For each {s1, s2,..., sk} in F Do Pour (k) 1: SetToZero( s1 ) 2: SetToZero( s2 )... k: SetToZero( sk ) Unite EndFor; MaxCountOfOnes

8. 7/9/99CEC'998 Pour(2) SetToZero(b) SetToZero(c) 001,101 010,100 SetToZero(a) SetToZero(b) 011 101 Pour(2) Example Initialize: 111 a b c MaxCountOfOnes: 2 001,101,010,100

9. 7/9/99CEC'999 3. Biomolecular Implementation u DNA modification enzymes –how to write on molecules u DNA plasmid –use of bacteria and blue/white selection

10. 7/9/99CEC'9910 Write on molecules u Restriction enzyme –cuts DNA at a specific subsequence (site) 5’-TATCGA-3’ 3’-ATAGCT-5’ ↓ Hind III 5’-T ATCGA-3’ 3’-ATAGC T-5’ u Circular DNA + modification enzymes –Bit =1 (site exists), =0 (no site)

11. 7/9/99CEC'9911 Cut/fill/paste 5’-TATCGA-3’ Bit=1, circular 3’-ATAGCT-5’ cut↓ restriction enzyme 5’-T ATCGA-3’ linear 3’-ATAGC T-5’ fill↓ DNA polymerase 5’-TATCG ATCGA-3’ 3’-ATAGC TAGCT-5’ paste↓ DNA ligase 5’-TATCGATCGA-3’ 3’-ATAGCTAGCT-5’ Bit=0, circular

12. 7/9/99CEC'9912 Cloning with DNA plasmid u DNA plasmid –circular, double stranded –set of unique sites »multiple cloning site (MCS) u transform to bacteria –useful genes »antibiotics resistance (ex.amp r ) »coloring matters (b-galactosidase) amp r  -galactosidase MCS NotI XbaI SpeI BamHI XmaI PstI EcoRI EcoRV HindIII... 5’-GCGGCCGCTCTAGAACTAGTGGATCCCCCGGGCTGCAGGAATTCGATATCAAGCTTATCGAT-3’ 3’-CGCCGGCGACATCTTGATCACCTAGGGGGCCCGACGTCCTTAAGCTATAGTTCGAATAGCTA-5’

13. 7/9/99CEC'9913 Genetic code translation u Genetic code –translated into a series of amino acids by groups of 3 base pairs (codon) u Reading frame –3 different meanings ex)5’-GCTCTAGAACTAGTGGATCCCCCGGGCTGCAGGAATTCGATATC A L E L V D P P G C R N S I.............. (under construction)

14. 7/9/99CEC'9914 Blue / white selection u initial DNA plasmid express  -galactosidase gene → blue ↓ u 1st cut/fill/paste +4bp ⇒ reading frame shift → white ↓ u 2nd cut/fill/paste +8bp ⇒ reading frame still shift → white ↓ u 3rd cut/fill/paste +12bp ⇒ readinf frame restored → blue »useful as a debugging tool

15. 7/9/99CEC'9915 Blue/white example

16. 7/9/99CEC'9916 Preliminary results XbaI BamHI HindIII pBSK GCTCTAGAACTAGTGGATCCCCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATACCGTCG A L E L V D P P G C R N S I S S L S I P S [H] GCTCTAGAACTAGTGGATCCCCCGGGCTGCAGGAATTCGATATCAAGCTAGCTTATCGATACC A L E L V D P P G C R N S I S S stop [HB] GCTCTAGAACTAGTGGATCGATCCCCCGGGCTGCAGGAATTCGATATCAAGCTAGCTTATCGA A L E L V D R S P G L Q E F D I K L A Y R [HBX] GCTCTAGCTAGAACTAGTGGATCGATCCCCCGGGCTGCAGGAATTCGATATCAAGCTAGCTTA A L A R T S G S I P R A A G I R Y Q A S L sampleblue / white accuracy [H] 4 / 4087% [HB] 3 / 8096% [HBX] 97 / 1785% SetToZero Hind III ->BamH I ->Xba I

17. 7/9/99CEC'9917 Example [HB] (+8, white) a=0 (SpeI) b=0 (XhoI) b=0 (XhoI) c=0 (XbaI) mix; +12 & +16 (solution = +12, white) a b c 0 +4 +8 +12 under construction

18. 7/9/99CEC'9918 4. Discussion u Advantages as DNA computing –start with one DNA plasmid »no custom DNA for individual problem –amplify in bacteria »blue/white selection as debugging tool »preserving the distribution of DNA plasmids

19. 7/9/99CEC'9919 5. Conclusion u Molecular Memory –Aqueous Algorithm »general framework to use molecular memory –Cut/fill/paste »laboratory implementation u Further issues –scale up & speed up –new algorithm fits bacteria

20. 7/9/99CEC'9920 International Connection Binghamton University (USA) Leiden University (Netherlands) Tokyo Institute of Technology (Japan) Aqueous Computing

21. 7/9/99CEC'9921 Acknowledgement u Xia Chen & Shalini Aggarwal in S.Gal Laboratory at Binghamton University u NSF CCR-9509831 u DARPA/NSF CCR-9725021 u JSPS-RFTF 96100101 u LCNC at Leiden University