Reaction discovery enabled by DNA-templated synthesis and in vitro selection

1. 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, 2004 Presented by Seok, Ho-Sik BioIntelligence Lab

2. 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 BioIntelligence Lab

3. 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 BioIntelligence Lab

4. 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 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 BioIntelligence Lab

5. Linker Biotin + disulphide bond How? – Reaction & Separation Watson-Crick pairing • The mixture n x m discrete pairs of substrates • Separation using avidin affinity • Detection by PCR (polymerase chain reaction) BioIntelligence Lab

6. Bond forming BioIntelligence Lab

7. 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 BioIntelligence Lab

8. 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 Separation in detail • Separation BioIntelligence Lab

9. 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 affinity • When these two molecules are in the same solution, they will bind with such high affinity that the binding is essentially irreversible BioIntelligence Lab

10. Purification using Avidin-Biotin reaction Affinity column Wash off proteins that do not bind Proteins sieve through matrix of affinity beads BioIntelligence Lab

11. How? – Detection BioIntelligence Lab

12. 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 BioIntelligence Lab

13. How? – Detection of putative reactions BioIntelligence Lab

14. Detection of putative reactions in detail Denaturing PolyaArylamide Gel Electrophoresis (PAGE) analysis Matrix Assisted Laser DesorptionIonization–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 BioIntelligence Lab

15. 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 BioIntelligence Lab