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Reaction discovery enabled by DNA-templated synthesis andin vitro selection

Matthew W. Kanan, Mary M. Rozenman, Kaori Sakurai,Thomas M. Snyder & David R. Liu

Nature, vol.431, 2004Presented by Seok, Ho-Sik

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What they did?

A reaction discovery approach that uses DNA-templated o

rganic synthesis and in vitro selection to simultaneously

evaluate many combination of different substrates for bo

nd-forming reactions in a single solution

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How? – Strand Preparation Organizing complex substrate mixtures into discrete

pairs Discrete pairs must react without affecting the reactivity of

the other substrate pairs

Index

Each substrate in pool A is covalently linked to the 5` end

Each substrate in pool B is covalently linked to the 3` end

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Strand Preparation in detail Recent developments in DNA-templated organic synthesi

s indicate that DNA annealing can organize many substrates in a single solution into DNA sequence-programmed pairs

Two pools of DNA-linked substrates, with n substrates in pool A and m substrates in pool B Each substrate in pool A is covalently linked to the 5` end of a set

of DNA oligonucleotides containing one ‘coding region’ (uniquely identifying that substrate) and one of m different ‘annealing regions’

Each of the m substrates in pool B is attached to the 3` end of an oligonucleotide containing a coding region that uniquely identifies the substrate and complements one of the m annealing regions in pool A

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How? – Reaction & Separation

The mixture n x m discrete pairs of substrates

Watson-Crick pairing

Separation using avidin affinity Detection by PCR (polymerase chain reaction)

Linker

Biotin + disulphide bond

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Bond forming

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Reaction in detail

Role of Watson-Crick base pairing When pools A and B are combined in a single aqueous solution,

Watson–Crick base pairing organizes the mixture into n x m discre

te pairs of substrates attached to complementary sequences

Only substrates linked to complementary oligonucleotides experie

nce effective molarities in the millimolar range

Possibility of interference by the DNA structure Minimized by using long and flexible substrate–DNA linkers

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Separation in detail Separation

Incubation under a set of chosen reaction conditions

Cleavage of the disulphide bonds

Only pool A sequences encoding bond formation between a pool A and pool B substrate remain covalently linked to biotin

Streptavidin affinity selection of the resulting solution separates biotinylated from non-biotinylated sequences

PCR

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Avidin-Biotin in detail Avidin/streptavidin-biotin systems are particularly useful

as a bridging or sandwich system in association with antigen-antibody interactions

Biotin and Avidin Biotin: a small organic molecule found in every cell Avidin: a much larger protein that binds biotin with a very high affi

nity When these two molecules are

in the same solution, they will bind with such high affinity that the binding is essentially irreversible

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Affinity column

Proteins sieve through matrix of affinity beads

Wash off proteins that do not bind

Purification using Avidin-Biotin reaction

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How? – Detection

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Detection in detail Capturing

Sequences encoding bond-forming substrate pairs were captured with streptavidin-linked magnetic particles

Amplification Sequences encoding bond-forming substrate pairs were amplified by PCR

with a DNA primer labeled with the cyanine fluorophore Cy3 Comparison

Aliquot of the pool A sequences before selection was amplified by PCR with a Cy5-labelled primer

Scoring The ratios of Cy3 (green) to Cy5 (red) fluorescence for all array locations

were calculated and ordered by rank, and spots with green/red fluorescence ratios significantly higher than the majority of spots (in the experiments below, ratios above 1.5) were considered to be positive

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How? – Detection of putative reactions

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Detection of putative reactions in detailDenaturing PolyaArylamide Gel Electrophoresis (PAGE) analysis

Matrix Assisted Laser Desorption Ionization–Time-of-Flight (MALDI–TOF) mass spectrometry

PAGE Comparison of strand positions

MALDI-TOF Once inside the ionisation source the

sample molecules are ionised, because ions are easier to manipulate than neutral molecules

These ions are extracted into the analyser region of the mass spectrometer where they are separated according to their mass-to-charge ratios

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DNA computing and the reaction discovery Another method for providing strands

Another method for selecting strands In addition to affinity, we can use cleavage of bonds

Expensive but precise way of detecting MALDI–TOF mass spectrometry

Possibility of advanced DNA computing Practical limitation of 10k

Possibility of DNA as a just template or catalyst

Using product of DNA-templated reaction for computing