Towards Reverse Transcription with an Expanded Genetic Alphabet

Area of Science:

• Synthetic biology
• Molecular biology
• Biochemistry

Background:

• Unnatural base pairs (UBPs) significantly expand the genetic alphabet beyond the natural DNA/RNA bases.
• UBPs hold potential for evolving novel aptamers and ribozymes through in vitro selection (SELEX).
• The application of UBPs in reverse transcription (RTC), a crucial step in RNA-based SELEX, remains largely unexplored.

Purpose of the Study:

• To investigate the compatibility of the Romesberg NaM:TPT3 UBP with reverse transcription reactions.
• To evaluate the performance of commercially available reverse transcriptases (RTs) with UBPs.
• To assess the utility of RTs in verifying UBP presence and abundance in RNA.

Main Methods:

• Testing five commercial reverse transcriptases (RTs) with RNA templates containing the NaM:TPT3 UBP.
• Analyzing RT pausing and primer extension efficiency at UBP sites.
• Quantifying full-length cDNA synthesis from UBP-containing RNA templates.

Main Results:

• Most tested RTs predominantly pause at the unnatural rTPT3 nucleotide, unable to incorporate the dNaM substrate.
• This pausing allows for the verification of UBP position and estimation of abundance in RNA.
• Primer extension from templates containing rNaM showed significantly higher full-length cDNA synthesis.

Conclusions:

• The NaM:TPT3 UBP presents challenges for most current RTs, primarily due to pausing at rTPT3.
• RT pausing at UBPs can be leveraged for detecting and quantifying unnatural bases in RNA.
• Certain RTs demonstrate potential for accommodating an expanded genetic alphabet incorporating NaM:TPT3 UBPs.