Minimizing amplification bias during reverse transcription for in vitro selections.

Systematic evolution of ligands through exponential enrichment (SELEX) is widely used to identify functional nucleic acids, such as aptamers and ribozymes. Ideally, selective pressure drives the enrichment of sequences that display the function of interest (binding, catalysis, etc.). However, amplification biases from reverse transcription can overwhelm this enrichment and leave some functional sequences at a disadvantage, with cumulative effects across multiple rounds of selection. Libraries that are designed to include structural scaffolds can improve selection outcomes by sampling sequence space more strategically, but they are also susceptible to such amplification biases, particularly during reverse transcription. Therefore, we tested five reverse transcriptases (RTs)-ImProm-II, Marathon RT (MaRT), TGIRT-III, SuperScript IV (SSIV), and BST 3.0 DNA polymerase (BST)-to determine which enzymes introduced the least bias. We directly compared cDNA yield and processivity for these enzymes on RNA templates with varying degrees of structure under various reaction conditions. In these analyses, BST exhibited excellent processivity, generated large quantities of the full-length cDNA product, displayed little bias among templates with varying structure and sequence, and performed well on long, highly structured viral RNAs. Additionally, six RNA libraries containing either strong, moderate, or no incorporated structural elements were pooled and competed head-to-head in six rounds of an amplification-only selection without external selective pressure using either SSIV, ImProm-II, or BST during reverse transcription. High-throughput sequencing established that BST maintained the most neutral enrichment values, indicating low interlibrary bias over the course of six rounds, relative to SSIV and ImProm-II, and it introduced minimal mutational bias.

Post-transcriptional capping generates coenzyme A-linked RNA.

NAD can be inserted co-transcriptionally via non-canonical initiation to form NAD-RNA. However, that mechanism is unlikely for CoA-linked RNAs due to low intracellular concentration of the required initiator nucleotide, 3'-dephospho-CoA (dpCoA). We report here that phosphopantetheine adenylyltransferase (PPAT), an enzyme of CoA biosynthetic pathway, accepts RNA transcripts as its acceptor substrate and transfers 4'-phosphopantetheine to yield CoA-RNA post-transcriptionally. Synthetic natural (RNAI) and small artificial RNAs were used to identify the features of RNA that are needed for it to serve as PPAT substrate. RNAs with 4-10 unpaired nucleotides at the 5' terminus served as PPAT substrates, but RNAs having <4 unpaired nucleotides did not undergo capping. No capping was observed when the +1A was changed to G or when 5' triphosphate was removed by RNA pyrophosphohydrolase (RppH), suggesting the enzyme recognizes pppA-RNA as an ATP analog. PPAT binding affinities were equivalent for transcripts with +1A, +1 G, or 5'OH (+1A), indicating that productive enzymatic recognition is driven more by local positioning effects than by overall binding affinity. Capping rates were independent of the number of unpaired nucleotides in the range of 4-10 nucleotides. Capping was strongly inhibited by ATP, reducing CoA-RNA production ~70% when equimolar ATP and substrate RNA were present. Dual bacterial expression of candidate RNAs with different 5' structures followed by CoA-RNA CaptureSeq revealed 12-fold enrichment of the better PPAT substrate, consistent with in vivo CoA-capping of RNA transcripts by PPAT. These results suggest post-transcriptional RNA capping as a possible mechanism for the biogenesis of CoA-RNAs in bacteria.