Cell and Microbial Engineering Laboratory Solution of a 20-Variable 3-SAT Problem on a DNA Computer Ravinderjit S. Braich, Nickolas Chelyapov, Cliff Johnson, Paul W. K. Rothemund, and Leonard Adleman Science vol April 2002 Summarized by Jiyoun Lee

Cell and Microbial Engineering Laboratory Introduction  The Boolean formula  20 variable with 24-clause 3-conjunctive normal form (3-CNF) formula,    was designed to have a unique satisfying truth assignment  Sticker model  Mix and split for half-library generation  Polymerase extension method for full-length library generation  Graduate PCR to read the answer

Cell and Microbial Engineering Laboratory Sticker model  Sticker model  Library + Sticker  Operations  Combine  Separation  Setting  Cleaning  Separation based on subsequence  use only  Application of stickers  Random access memory that requires no strand extension, uses no enzyme, and (at least in theory) its materials are reusable

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Separation operation  Separation using Acrydite TM phosphoamidite for modifying DNA molecules at 5’-end during chemical synthesis  Covalently linking the probes to the gel matrix  Gives one benefits of solid-support-based system while still remaining characteristics of a solution-based system  Separation  Oligonucleotide probes immobilized in polyacrylamide gel-filled glass modules  Capture with immobilized probes and release at a higher temperature

Cell and Microbial Engineering Laboratory Attachment of oligonucleotides to solid  Various methods are available to attach oligonucleotides to solid surfaces such as microarray slides, microtiter plates or magnetic beads, including:  Biotin-oligo non-covalently complexed with Streptavidin.  SH-oligo covalently linked via a disulfide bond to a SH-surface.  Amine-oligo covalently linked to an activated carboxylate or an aldehyde group.  Phenylboronic acid (PBA)-oligo complexed with salicylhydroxamic acid (SHA)

Cell and Microbial Engineering Laboratory Acrydite TM  Enables covalent attachment of oligonucleotides and other macromolecules to surfaces via acrylic linkages.  An oligonucleotide derivatized with Acrydite group can polymerize with acrylamide monomer to form polyacrylamide or can react with thiol or silane surfaces. This chemistry is also compatible for attachment to polymer surfaces.  2D  3D immobilization

Cell and Microbial Engineering Laboratory An acrylic acid group can be directly attached to the 5'-end of an oligonucleotide (with a 6-carbon linker arm ) at the time of synthesis using Acrydite, an acrylic- phosphoramidite developed by Mosaic Technologies

Cell and Microbial Engineering Laboratory The library I  X k T, X k F 15 base value sequences, 2 N library strands  Constraints  Library sequences contain only A, T, C  less secondary structure  All library and probe sequences have no occurrence of 5 or more consecutive identical nucleotides  Every probe sequence has at least 4 mismatches with all 15 base alignment of any library sequence  Every 15 base subsequence of a library sequence has at least 4 mismatches with all 15 base alignment of itself or any library sequence  No probe sequence has a run of more than 7 matches with any 8 base alignment of any library sequence  No library sequence has a run of more than 7 matches with any 8 base alignment of itself or any other library sequence  Every probe sequence has 4, 5, or 6 Gs in its sequence  Discourage intra- and interlibrary strand hybridization and unintended probe- library strand hybridization

Cell and Microbial Engineering Laboratory The library II  X k Z, 5’-end Acrydite-modified oligonucleotides  Used as probes  Synthesis of long molecules  Synthesis of two ‘half-libraries’ x 0 through x 10 (left half-library), x 11 through x 20 (right half-library)  Half-libraries: a mix-and split combinatorial synthesis technique was used  The 300-oligomer (300-mer) full library was created from the two half- libraries using a polymerase extension method

Cell and Microbial Engineering Laboratory Mix and split  Combinatorial DNA library construction (half-library)  During synthesis

Cell and Microbial Engineering Laboratory Polymerase extension method  Assembly PCR method for the synthesis of long DNA sequences from large numbers of oligonucleotides  Does not rely on DNA ligase but instead relies on DNA polymerase to build increasingly longer DNA fragments during the assembly process  Derived from DNA shuffling

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Left half (1~10) 150 merRight half (11~20) 150 mer X 11 X 10 5’5’ 3’3’ 5’5’ 3’3’ 5’5’ 3’3’ 10 pmole 2 pmole each  Final volume 20  l in 1X T4 DNA ligase buffer, incubate at RT for 2 hrs Mixture 0.5  l Primer: X 1 T, X 1 F, Acrydite-modified ~X 20 T, ~X 20 F 1 ml aliquot, PCR again Band extraction, creat stock solution

Cell and Microbial Engineering Laboratory

The computer and the computational protocol  Step 1: Insert the library module into the hot chamber of the electrophoresis box and the first clause module into the cold chamber of the box. Begin electrophoresis.  Step 2: Remove both modules from the box. Discard the module from the hot chamber. Wash the box and add new buffer. Insert the module from the cold chamber into the hot chamber and the module for the next clause into the cold chamber. Begin electrophoresis.  Step 3: Repeat Step 2 for each of the remaining 22 clauses.  Step 4: Extract the answer strands from the final clause module, PCR- amplify, and “read” the answer.

Cell and Microbial Engineering Laboratory 0.5cm thick plexiglass Probe layer (releasing) Probe layer (capturing)

Cell and Microbial Engineering Laboratory A clause module 4.5 cm 3.2 cm

Cell and Microbial Engineering Laboratory Detection of the answer  Graduate PCR

Cell and Microbial Engineering Laboratory

Capture-release efficiency