Project Grant R43GM150322

ELECTRONICALLY ADDRESSABLE DNA ASSEMBLY - THE ABILITY TO ROUTINELY SYNTHESIZE ENTIRE GENOMES IS ONE OF THE GREAT REMAINING TECHNICAL ASPIRATIONS OF THE SYNTHETIC BIOLOGY REVOLUTION. THE COMPLEXITY OF DESIGNING THE ANNEALING REACTIONS USED BY EXISTING ASSEMBLY WORKFLOWS IS A FUNDAMENTAL BARRIER TO GENOME-SCALE DNA PRODUCTION. THERE IS A PRACTICAL LIMIT TO THE NUMBER OF DISTINCT SEQUENCES WHICH CAN RELIABLY SELF-ASSEMBLE INTO THE DESIRED CONSTRUCT IN A SINGLE STEP, AND TRADITIONAL ASSEMBLY REACTIONS ARE TYPICALLY LIMITED IN SCALE TO TENS OR HUNDREDS OF OLIGONUCLEOTIDES. CURRENT PARALLEL SYNTHESIS TECHNOLOGIES CAN GENERATE POOLS OF OVER 1 MILLION OLIGONUCLEOTIDES, WHICH IS ENTIRELY INADEQUATE FOR A GENOME-SCALE CONSTRUCTION, YET FAR MORE COMPLEX THAN CAN BE RELIABLY ASSEMBLED IN A SINGLE REACTION. DESPITE SUBSTANTIAL INTEREST IN IMPROVING OLIGONUCLEOTIDE SYNTHESIS THROUGHPUT, SUCH TECHNOLOGIES CANNOT BE LEVERAGED FULLY WITHOUT ACCOMPANYING MEANS OF GUIDING THEIR ASSEMBLY. WE HAVE MADE ADVANCEMENTS TO GENERATE OLIGONUCLEOTIDES AT A THROUGHPUT 100 ANY PUBLISHED SYSTEM. THE PROPOSED STUDIES WORK TO TRANSLATE THIS ADVANCE (UP TO 100 ENHANCEMENT) TO THE THROUGHPUT OF GENE ASSEMBLY. THE UNDERLYING CONCEPT IS TO PERFORM DNA SYNTHESIS ON A SUBSTRATE CAPABLE OF CONTROLLING THE LOCALIZATION OF THE OLIGONUCLEOTIDES DURING THEIR SUBSEQUENT ASSEMBLY. IF SUCCESSFUL, THE APPROACH WOULD ADDRESS A FUNDAMENTAL SCALABILITY MISMATCH BETWEEN OLIGONUCLEOTIDE SYNTHESIS AND ASSEMBLY, ENABLING A NEW GENERATION OF GENE SYNTHESIS TECHNOLOGIES CAPABLE OF EXCEEDING THE SCALE OF THE HUMAN GENOME WITH A SINGLE SYNTHESIS RUN. THIS PROJECT MAY LEAD TO A 100X PARALLELIZATION INCREASE AND 100X COST DECREASE, AND POTENTIALLY INCREASE ASSEMBLY ACCURACY.