Nucleotide analogues play important roles in biological pathways, such as DNA replication, and as essential enzyme cofactors. Bioorganic chemists, by synthesizing and examining the properties of novel nucleotide derivatives, have helped advance such chemistry of life processes areas as DNA sequencing and labeling, real time molecular tracking in living cells, developing mutagenesis models, oligonucleotide structural probes, and clinical therapeutic agents. Molecular recognition of unnatural nucleotides by DNA polymerases is essential to promote the incorporation of chemical modifications into DNA molecule by polymerase chain reaction, both in vitro and in vivo. However, the major drawback of nucleotides with large, specific modifications is the lack of recognition by natural enzymes, especially if the modification blocks the 3’-hydroxyl or interferes with Watson-Crick base pairing. This often represents serious obstructions, in particular for the use of traditional nucleotide DNA synthesis terminators in live cell DNA sequencing by synthesis. In the course of mentored research, I and my co-workers have identified a modification for nucleobases that results in enhanced incorporation efficiency of the unnatural nucleotides into a partial double helix DNA template compared to their natural counterparts. Depending on the structure of the modifying group, incorporation of the nucleotide analog may result either in continued DNA elongation or termination of further DNA synthesis upon a single incorporation event. During my independent research, I have extended the initial application of the newly developed methodology from the in-vitro DNA sequencing-by-synthesis to examining the corresponding base-modified nucleotides as agents for DNA synthesis termination in living cells. My findings demonstrate that further development of these novel base-modified nucleotides can provide new molecular tools with various applications in biochemistry, molecular biology, and drug discovery, ranging from investigation of intracellular DNA synthesis, structure, and function to the development of novel chemotherapeutic agents against cancers and viral infections.

Novel base-modified nucleotides terminating DNA synthesis and their applications in chemistry of life processes

249th National Meeting (2015)

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