Site-specific labeling of DNA and RNA using an efficiently replicated and transcribed class of unnatural base pairs

Site-specific labeling of enzymatically synthesized DNA or RNA has many potential uses in basic and applied research, ranging from facilitating biophysical studies to the in vitro evolution of functional nucleic acids and the construction of various nanomaterials and biosensors. As part of our efforts to expand the genetic alphabet, we have developed a class of unnatural base pairs, exemplified by d5SICS-dMMO2 and d5SICS-dNaM, which are efficiently replicated and transcribed, and which may be ideal for the site-specific labeling of DNA and RNA. Here, we report the synthesis and analysis of the ribo- and deoxyribo-variants, (d)5SICS and (d)MMO2, modified with free or protected propargylamine linkers that allow for the site-specific modification of DNA or RNA during or after enzymatic synthesis. We also synthesized and evaluated the alpha-phosphorothioate variant of d5SICSTP, which provides a route to backbone thiolation and an additional strategy for the postamplification site-specific labeling of DNA. The deoxynucleotides were characterized via steady-state kinetics and PCR, while the ribonucleosides were characterized by the transcription of both a short, model RNA as well as full length tRNA. The data reveal that while there are interesting nucleotide and polymerase-specific sensitivities to linker attachment, both (d)MMO2 and (d)5SICS may be used to produce DNA or RNA site-specifically modified with multiple, different functional groups with sufficient efficiency and fidelity for practical applications.

Références

Titre
Site-specific labeling of DNA and RNA using an efficiently replicated and transcribed class of unnatural base pairs
Type de publication
Article de revue
Année de publication
2011
Revue
Journal of the American Chemical Society
Volume
133
Pagination
19878-88
Soumis le 5 juillet 2018