DNA computing using single-molecule hybridization detection

1. DNA computing using single-molecule hybridization detection K.A.Schmidt, C.V.Henkel, G.Rozenberg, and H.P.Spaink, NAR , vol. 32, no. 17, Sep. 2004, pp. 4962-4968. Summarized by Byoung-Hee Kim 2004.11.20

2. Overview • Problem: 3-SAT • Algorithm: blocking algorithm • Experiment • Synthesis • Hybridization • Detection • Destroy • Filtering, marking • Blocking (Direct inactivation of those molecules that are not a solution) Single molecule detection by FCS, dual-color FCCS

3. formula clause Problem • 4-variable 3-SAT problem • 24 possible solutions • Falsifying one clause is sufficient for falsifying the complete formula • Blocker: falsifying case

4. formula clause Sequence design • General structure • (5’)start – a b c d – stop(3’) • Start : CTTGCA • Stop : TTGCAC • Bit=0 : ATCACC • Bit=1 : GTCTGA • Library:16(24) different oligonucleotides of 36bp each • Blockers: designed to be complementary to the library oligos

5. clause A clause B clause C clause D Overall encoding scheme

6. Library molecule blocker Blocking algorithm • (Rozenberg and Spaink, 2003) • Basic idea : Direct inactivation of those molecules that are not a solution Build library Add blockers (Blocker construction can be automated) • Special prefix and suffix • Use PNA (peptide nucleic acid) blockers to prevent blocked items being amplified PCR • IF (PCR success) THEN (there are solutions) • Detection by visual inspection (E.g. DNA-specific dyes) Detection

7. Fluorescent labeling Cy5 5’ 3’ Library molecule blocker 3’ 5’ Rhodamine green Blocking: detect hybridized molecule, or dsDNA DNA 사용량 FRET Heteroduplex migration assay Enzymatic mismatch recognition FCS μM nM

8. Detection volume, <0.5 fℓ FCS • FCS (fluorescence-correlation spectroscopy) • Studies fluorescence fluctuations caused by a single molecule diffusing in the focal detection volume (figures are from http://www.probes.com/handbook/boxes/1571.html)

9. Correlator FCCS • FCCS (fluorescence cross-correlation spectroscopy) • the number of doubly labeled particles(Ncc) can be calculated Laser1 488 nm Laser2 633 nm Fitted curve fluorescent signal Beam splitter Avalanche photodiode

10. b mismatch L03 A0,A1 Perfect match Perfect match c L04 L11 B0 A0,A1 mismatch Perfect match 4 bp mismatch L04 L05 B0,B1,C1 B0,B1,C1 L05 B0 Hybridization detection at the level of single DNA molecules a • All oligonucleotide concentrations : 10 nM a: single blocker with two library molecules b: addition of two types of blocker c: addition of three types of blocker ※ when using four different blocker, the amplitudes were too low

11. clause A Blocker A clause B Blocker B clause C Blocker C clause D Blocker D Complete computation • FIND-SAT • Find out satisfiable instances for 3-SAT problem • Hybridize with each library molecule (L00~L15) • Total trial # : 24 × (#clauses) × (3~4 repeat)

12. Complete computation - Result b a c

13. Paradigm Blocking algorithm Experiment Single molecular detection by FCS Overview revisited No PCR Solved problem: 3-SAT

14. Discussion

15. meaning Experimental meaning promise • The first example for the utilization of single-molecule techniques for DNA computing • Fluorescence cross-correlation spectroscopy was employed • In contrast to all previous approaches, requires • -neither large quantities of DNA for detection • -nor destroy the output molecules • Promise for extending the size of the libraries • Considerable improvement compared to the FRET assay • Less prone to experimental errors Bloking algorithm & Silgne molecule detection • Combination with LOC (Lab-on-a-chip) => enhance the parallelism of the computation • Combination with microfluidics systems => high-throughput hybridization detection • Utilization of universal nucleotides => may decrease the number of blocker molecules • The amount of DNA is much reduced => by four orders of magnitude => the search space may be increased 10,000 times • Much faster • Easily combined with high-throughput screening • attractive for implementing evolutionary algorithms with DNA • The selection step is difficult, very high background of ‘wrong’ molecules • ‘Single molecule technique’ and ‘non-destructive character’ has the potential to overcome these problem Future works Vs gel electrophoresis Evolutionary DNA computing

16. References – blocking algorithm [1] Rozenberg,G. and Spaink,H. (2003) DNAcomputing by blocking. Theor.Comp. Sci., 292, 653–665. [2] Schmidt,K.A., Henkel,C.V., Rozenberg,G. and Spaink,H.P. (2002) Experimental aspects of DNA computing by blocking: use of fluorescence techniques for detection. In Kraayenhof,R., Visser,A.J.W.G. and Gerritsen,H.C. (eds), Fluorescence Spectroscopy, Imaging and Probes—New Tools in Chemical, Physical and Life Science. Springer-Verlag, Heidelberg, Germany, pp. 123–128. [3] Henkel,C.V., Rozenberg,G. and Spaink,H.P. (2004) Application of mismatch detection methods in DNA computing. In Ferreti,C., Mauri,G. and Zandron,C. (eds), DNA10, 10th International Meeting on DNA Computing, Preliminary Proceedings. Universita di Milano-Bicocca, pp. 183–192.

17. References - FCS [1]Madge,D., Webb,W.W. and Elson,E. (1972) Thermodynamic fluctuations in a reacting system – measurement by fluorescence correlation spectroscopy. Phys. Rev. Lett., 29, 705–708. [2]Eigen,M. and Rigler,R. (1994) Sorting single molecules: application to diagnostics and evolutionary biotechnology. Proc. Natl Acad. Sci. USA, 91, 5740–5747.