Reverse transcription as key step in RNA in vitro evolution with unnatural base pairs.

Unnatural base pairs (UBPs) augment the chemical diversity of artificial nucleic acids and can thus enable the generation of new aptamers and catalytic nucleic acids by in vitro selection. However, owing to a lack of methodologies, the reverse transcription of UBPs, a key step in RNA aptamer selection, has not been sufficiently characterized. Here, we present a series of versatile assays to investigate the reverse transcription of the TPT3:NaM base pair as a representative for hydrophobic unnatural base pairs. We determine the fidelity and retention of the UBP for four different reverse transcriptases (RT) in the context of RNA in vitro evolution. The retention of the TPT3:NaM pair during the RNA in vitro selection process was investigated using a novel click-chemistry based electromobility shift assay. Real-time monitoring of reverse transcription kinetics revealed considerable differences in polymerase activity processing the TPT3:NaM base pair. Our findings identified SuperScript IV RT as the most efficient RT for processing the TPT3:NaM pair. Our approach can be applied universally to study newly developed UBPs, not only at the reverse transcription level, but also during PCR and in vitro transcription.

Kinetic FRET Assay to Measure Binding-Induced Conformational Changes of Nucleic Acids.

The interaction of small molecules or proteins with RNA or DNA often involves changes in the nucleic acid (NA) folding and structure. A biophysical characterization of these processes helps us to understand the underlying molecular mechanisms. Here, we propose kinFRET (kinetics Förster resonance energy transfer), a real-time ensemble FRET methodology to measure binding and folding kinetics. With kinFRET, the kinetics of conformational changes of NAs (DNA or RNA) upon analyte binding can be directly followed via a FRET signal using a chip-based biosensor. We demonstrate the utility of this approach with two representative examples. First, we monitored the conformational changes of different formats of an aptamer (MN19) upon interaction with small-molecule analytes. Second, we characterized the binding kinetics of RNA recognition by tandem K homology (KH) domains of the human insulin-like growth factor II mRNA-binding protein 3 (IMP3), which reveals distinct kinetic contributions of the two KH domains. Our data demonstrate that kinFRET is well suited to study the kinetics and conformational changes of NA-analyte interactions.