A simple in vitro assay to evaluate the incorporation efficiency of ribonucleotide analog 5' -triphosphates into RNA by human mitochondrial DNA-dependent RNA polymerase [PublishAheadOfPrint]

There is a growing body of evidence suggesting that some ribonucleoside/ribonucleotide analogs may be incorporated into mitochondrial RNA by human mitochondrial DNA-dependent RNA polymerase (POLRMT) and disrupt mitochondrial RNA synthesis. An assessment of incorporation efficiency of a ribonucleotide analog 5' -triphosphate by POLRMT may be used to evaluate potential mitochondrial toxicity of the analog early in the development process. In this report, we provide a simple method to prepare active recombinant POLRMT. A robust in vitro nonradioactive primer extension assay was developed to assay the incorporation efficiency of ribonucleotide analog 5' -triphosphates. Our results showed that many ribonucleotide analogs, including some antiviral compounds currently in various pre-clinical or clinical development stages, can be incorporated into newly synthesized RNA by POLRMT, and that the incorporation of some of them can lead to chain termination. The discrimination values (D values) of ribonucleotide analog 5' -triphosphates over natural ribonucleotide triphosphates (rNTPs) were measured to evaluate the incorporation efficiency of the ribonucleotide analog 5' -triphosphates by POLRMT. Discrimination values of natural rNTPs in the condition of misincorporation by POLRMT were used as a reference to evaluate the potential mitochondrial toxicity of ribonucleotide analogs. We proposed the following criteria for potential mitochondrial toxicity of ribonucleotide analogs based on D values: a safe compound (D > 10⁵); a potentially toxic compound (10⁵ > D > 10⁴); and a toxic compound (D < 10⁴). This report provides a simple screening method that should assist investigators in designing ribonucleoside-based drugs having lower mitochondrial toxicity.

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