Points to Consider for Chemically Modified Nucleotides

In principle, all therapeutic oligonucleotides that enter cells will eventually be metabolized to individual monomeric forms and be subject to endogenous nucleotide salvage pathways and join endogenous nucleotide pools within any given cell. The actual extent and rate at which this occurs will rely 

DISCOVERY AND DEVELOPMENT STRATEGIES FOR SMALL INTERFERING RNAs 

3 Potential Effects on Transcription or Translation To complement direct polymerase intaaction assays, the potential effects of nonnatural monomeric metaboUles on transcription and translation processes should also be considered. Cell-free recombinant transcriplion/translation kits are commercially available (bacterial or mammalian origin) to enable the characterization of individual monomeric metabolites. In principle, any bad actor chemically modified nucleotide could be identified via alterations to overall RNA yield/integrity. Again, these assays could be conducted in competition-type formats (modified NTP spiked into endogenous NTP pool) mimicking relative exposure ratios in vivo. For translation, synthetic RNA templates could be synthesized that contain the chemical modification in question to see whether the presence of that modification in an RNA template alters overall protein yield. As an early hazard ID-type screen, any nucleotide modifications that behave as chain terminators or have some other inhibitory activity should be readily identified. 

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