Download Reaction discovery enabled by DNA

Reaction discovery enabled by
DNA-templated synthesis and
in vitro selection
Matthew W. Kanan,
Mary M. Rozenman,
Kaori Sakurai,
Thomas M. Snyder
& David R. Liu
Nature, vol.431, 2004
Presented by
Seok, Ho-Sik
BioIntelligence Lab
What they did?

A reaction discovery approach that uses DNA-templated
organic synthesis and in vitro selection to
simultaneously evaluate many combination of different
substrates for bond-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
synthesis indicate that DNA annealing can organize
many substrates in a single solution into DNA sequenceprogrammed pairs
Two pools of DNA-linked substrates, with n substrates
in pool A and m substrates in pool B

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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
Watson-Crick pairing

The mixture
Linker
n x m discrete pairs of substrates
Biotin +
disulphide
bond


Separation using avidin affinity
Detection by PCR (polymerase chain reaction)
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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
discrete pairs of substrates attached to complementary sequences

Only substrates linked to complementary oligonucleotides
experience 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

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Biotin: a small organic molecule found in every cell
Avidin: a much larger protein that binds biotin with a very high
affinity
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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Purification using Avidin-Biotin reaction
Affinity column
Wash off proteins
that do not bind
Proteins sieve through
matrix of affinity beads
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How? – Detection
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Detection in detail

Capturing

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Amplification

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Sequences encoding bond-forming substrate pairs were amplified by
PCR with a DNA primer labeled with the cyanine fluorophore Cy3
Comparison


Sequences encoding bond-forming substrate pairs were captured with
streptavidin-linked magnetic particles
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 detail
Denaturing PolyaArylamide Gel Electrophoresis (PAGE) analysis
Matrix Assisted Laser Desorption
Ionization–Time-of-Flight (MALDI–TOF) mass spectrometry

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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

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Expensive but precise way of detecting

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In addition to affinity, we can use cleavage of bonds
MALDI–TOF mass spectrometry
Possibility of advanced DNA computing

Practical limitation of 10k
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Possibility of DNA as a just template or catalyst

Using product of DNA-templated reaction for computing
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