Abstract

X-ray crystallography has been central in determining structure-function relationships for biomacromolecules, such as nucleic acids and proteins, leading to a greater understanding of biochemical processes. The major barriers to nucleic acid structure determination by X-ray crystallography are the processes of crystallization and phasing. One strategy to overcome the latter challenge involves replacement of oxygen with selenium in a nucleotide. In the past two decades, selenium has been incorporated into oligonucleotides at many different sites, each with their specific advantages and limitations. Preparation of selenium modified nucleic acids often requires the multi-step synthesis of specialized phosphoramidites incorporated into oligonucleotides by solid-phase synthesis. It would be desirable to simplify the synthetic process to prepare these selenium containing oligonucleotides, which may result in their greater accessibility for crystallographers engaged in the determination of high resolution structural studies of nucleic acid structures.
In one approach, a simplified, greener and more versatile methodology to incorporate selenium into a solid support-bound oligonucleotide at the O-4 position of thymidine was explored. A range of reaction and deprotection conditions were evaluated, with promise of this approach demonstrated when selenium incorporation was attempted at a 5’-end residue of an oligonucleotide, followed by deprotection using a sterically hindered base. Another approach investigated the preparation of an oligonucleotide with 2’-deoxy-6-selenoinosine, a derivative of interest which was inspired by the base pairing properties of 2’-deoxyinsoine towards other nucleobases. In the isolation and purification of this oligonucleotide, it was observed that the major product obtained was a non-complementary homodimer oligonucleotide connected by an interstrand diselenide bridge. Conditions to remove this cross-link were evaluated and expand the potential of selenium modified nucleic acids beyond X-ray crystallography for applications as a responsive nanomaterial.