DNA computing using DNA computing using single-molecule hybridization single-molecule hybridization

detectiondetection

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 Kim2004.11.20

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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 byFCS, dual-color FCCS

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Problem

4-variable 3-SAT problem 24 possible solutions

Falsifying one clause is sufficient for falsifying the complete formula

Blocker: falsifying case

).~(~)~()~(~)~( dcadbacbadcbF clauseformula

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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 oli

gos

).~(~)~()~(~)~( dcadbacbadcbF clauseformula

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Overall encoding scheme

).~(~)~()~(~)~( dcadbacbadcbF clause A clause B clause C clause D

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Blocking algorithm

(Rozenberg and Spaink, 2003) Basic idea : Direct inactivation of those molecules that are

not a solution

blocker

PCR

Detection

Build library

Add blockers

Library molecule

•Special prefix and suffix •Use PNA (peptide nucleic acid) blockers to prevent blocked items being amplified

•IF (PCR success) THEN (there are solutions)•Detection by visual inspection (E.g. DNA-specific dyes)

(Blocker construction can be automated)

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Fluorescent labeling

5’ 3’

3’ 5’

Library molecule

blocker

Cy5

Rhodamine green

Blocking: detect hybridized molecule, or dsDNA

FRETHeteroduplex migration assayEnzymatic mismatch recognitionFCS

μM

nM

DNA 사용량

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FCS

FCS (fluorescence-correlation spectroscopy) Studies fluorescence fluctuations caused by a single

molecule diffusing in the focal detection volume

Detection volume, <0.5 fℓ

(figures are from http://www.probes.com/handbook/boxes/1571.html)

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FCCS

FCCS (fluorescence cross-correlation spectroscopy) the number of doubly labeled particles(Ncc) can be calculated

fluorescent signal

Laser1488 nm

Laser2633 nm

Beam splitter

Avalanche photodiode

Correlator

Fitted curve

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Hybridization detection at the level of single DNA molecules

L04 B0

Perfect match

L05 B0

4 bp mismatch

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

b

c

L11 A0,A1

Perfect match

L03 A0,A1mismatch

L04 B0,B1,C1

Perfect match

L05 B0,B1,C1mismatch

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Complete computation

FIND-SAT Find out satisfiable instances for 3-SAT problem

).~(~)~()~(~)~( dcadbacbadcbF clause A clause B clause C clause D

Blocker A Blocker B Blocker C Blocker D

•Hybridize with each library molecule (L00~L15)

•Total trial # : 24 × (#clauses) × (3~4 repeat)

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Complete computation - Result

a b

c

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Overview revisited

No PCR

Solved problem:3-SAT

Blocking algorithm

Paradigm

Single molecular detection by FCS

Experiment

DiscussionDiscussion

Bloking algorithm &Silgne molecule

detection

meaning Experimental meaning promise

Future works Vs gel electrophoresis Evolutionary DNA computing

•The first example for the utilization of single-molecule techniques for DNA computing

•Fluorescence cross-correlation spectroscopy was employed

•Considerable improvement compared to the FRET assay

•Less prone to experimental errors

•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

•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

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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.

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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.