Project Description

The synthesis of nucleic acids with custom sequences is a very important tool in the research of biological systems. The prevailing strategy for DNA synthesis is the phosphoramidite strategy, which involves anhydrous solvents and acidic environments that are slightly harmful to DNA, causing small amounts of side reactions that limit the overall yield. This approach is not capable of producing DNA fragments over 200 bases long. One potential alternative strategy would involve enzyme catalysts to overcome the inefficiencies caused by side-reactions, though very little investigation has been conducted to this end, likely due to the increased expense of biologically derived components such as enzymes and deoxynucleoside triphosphates (dNTPs). The aim of this project is to develop a new DNA synthesis protocol using Terminal deoxynucleotidyl Transferase (TdT), a template-independent polymerase that nonspecifically adds nucleotides to 3’ ends, while applying recently developed technology to produce both TdT and dNTPs in a less expensive manner. This enzymatic approach has the potential to surpass traditional synthetic strategies in efficiency and yield.

The lack of reactivity of phosphates under standard chemical conditions stems partly from the charge on the anionic oxygens, which repel most nucleophilic attacks. Due to this inherent stability, enzymes are typically required for even simple phosphate ester manipulations. The primary challenge facing every chemical strategy for the synthesis of oligonucleotides is the formation of the phosphodiester bonds that string nucleosides together. Because these strategies must use activating tetrazole reagents to increase the reactivity of phosphates, side reactions are an unavoidable consequence. Despite this difficulty, an elegant solution employing phosphoramidite groups allows for the chemical synthesis of custom oligonucleotides up to a maximum length of 200 bases, for pennies per base.

Enzymatic strategies for custom oligonucleotide synthesis have been theorized but nonetheless remain understudied likely because of the expense of enzymes and dNTPs, which are both biologically derived. By implementing recent developments in the fields of protein expression/purification such as bacterial secretion[6] and non-chromatographic purification through precipitation tags, as well as experimental nucleotide 3’ acetylation strategies, we should be able to develop an inexpensive protocol for the production of TdT polymerase and introduction of 3’ blocking groups onto standard dNTPs.

The main DNA synthesis side reaction that raises concern is the depurination of nitrogenous bases from their respective ribose carbons, which occurs due to the acidic environment required for deblocking (detritylation). Another source of error is the inevitable presence of moisture in the anhydrous solvents, hydrolyzing the modified phosphates in solution. These errors are important to avoid because they detract cumulatively from the overall yield of final oligo product. We intend to demonstrate the simplicity of an enzymatic approach, utilizing base-labile blocking groups and neutral buffer environments to avoid depurination and other side-reactions.

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