Towards Reverse Transcription with an Expanded Genetic Alphabet

Abstract

Unnatural base pairs (UBPs) strikingly augment the natural genetic alphabet. The development of particular hydrophobic UBPs even allows insertion and stable propagation in bacteria. Those UBPs expand the chemical scope of DNA and RNA, and thus, could enable the evolution of novel aptamers or ribozymes by in vitro selection (systematic evolution of ligands by exponential enrichment, SELEX). However, the application of such UBPs in reverse transcription (rtc), which is a key step for RNA‐based SELEX, has not been reported so far. The implication of Romesberg's NaM:TPT3 base pair in rtc reactions is presented by testing five commercially available reverse transcriptases (RTs). The employed RTs predominantly pause at the site of the unnatural nucleotide rTPT3 not being able to accept the dNaM building block as a substrate. This allows verification of the unnatural base position in RNA and an estimation of their abundance. In contrast, primer extension from an rNaM‐containing template results in considerably more full‐length cDNA. Furthermore, RTs that could potentially be able to handle an expanded genetic alphabet based on NaM:TPT3 are presented.

Transcription and Reverse Transcription of an Expanded Genetic Alphabet In Vitro and in a Semisynthetic Organism

“…At the highest concentration of tRNA (25 ng/μL), reverse transcription of NaM or TPT3 templates in the presence of their corresponding unnatural deoxyribotriphosphate resulted in 88% and 44% full-length product, respectively. This corresponds well with values reported by Kath-Schorr et al of 90% and 33%, respectively, even though a different reverse transcriptase (MMLV) was used . Interestingly, the percentage of full length product increased to 97% and 92%, with the lower concentration of 0.5 ng/μL NaM or TPT3 tRNA template, respectively (Figure C, Table S1).…”

“…This corresponds well with values reported by Kath-Schorr et al of 90% and 33%, respectively, even though a different reverse transcriptase (MMLV) was used. 10 Interestingly, the percentage of full length product increased to 97% and 92%, with the lower concentration of 0.5 ng/μL NaM or TPT3 tRNA template, respectively (Figure 1C, Table S1).…”

“…In addition, while it is clear that different DNA polymerases, T7 RNA polymerase, and E. coli ribosomes productively recognize the UBP, the ability of reverse transcriptases, which mediate the only other common DNA/RNA transaction, has not been thoroughly explored, and the only available data suggests that they might not productively recognize the UBP. 10 To begin to examine these issues, we sought to develop an assay for UBP retention after sequential in vitro transcription (IVT) with T7 RNA polymerase and reverse transcription (RT) with the commercially available reverse transcriptases: SuperScript III, SuperScript IV, and AMV RT. Briefly, DNA containing the EGFP gene with dNaM or dTPT3 located at the position encoding the second nucleotide of codon 151 was PCR amplified and used as a template for IVT reactions, which were supplemented with the corresponding unnatural ribonucleoside triphosphate.…”

“…However, the lack of an assay to measure transcription fidelity has prevented the identification of the specific step that compromises fidelity. In addition, while it is clear that different DNA polymerases, T7 RNA polymerase, and E. coli ribosomes productively recognize the UBP, the ability of reverse transcriptases, which mediate the only other common DNA/RNA transaction, has not been thoroughly explored, and the only available data suggests that they might not productively recognize the UBP …”

From polymerase engineering to semi-synthetic life: artificial expansion of the central dogma

“…Recently, reverse transcription of RNA containing TPT3 or NaM has been investigated with several reverse transcriptases or DNAP mutant, including avian myeloblastosis virus (AMV) reverse transcriptase, Moloney murine leukemia virus (MMLV) reverse transcriptase, SuperScript II reverse transcriptase, SuperScript III reverse transcriptase, SuperScript IV reverse transcriptase, and an engineered Taq DNAP with reverse transcription activity, Volcano2G (V2G)153,154 . It was found that the UBP reverse transcription efficiencies of different reverse transcriptases were sharply different.…”