Fig. 2: UBP-based strategy for RNA fluorescent labeling facilitates TIRF-based smFRET measurements of flaviviral xrRNAs. | Nature Communications

Fig. 2: UBP-based strategy for RNA fluorescent labeling facilitates TIRF-based smFRET measurements of flaviviral xrRNAs.

From: Pseudoknot length modulates the folding, conformational dynamics, and robustness of Xrn1 resistance of flaviviral xrRNAs

Fig. 2

a Schematic representation of the TIRF-based smFRET analysis of the folding of flaviviral xrRNAs. The low-FRET (Lstate), intermediate-FRET (Istate), and high-FRET (Hstate) states correspond to the unfolded, partially folded, and folded states of xrRNA1, respectively. b Chemical structures of the parental NaM-TPT3 unnatural base pair and alkyne-modified TPT3 (rTPT3CO). c DNA templates of xrRNAs containing T7 promoter (shown in blue) and dNaM modification at the template strands were prepared by two-steps overlapping PCR, which was followed by in vitro transcription using rNTP mix supplemented with rTPT3COTP, allowing site-specific labeling of purified transcripts with azide-modified Cy5 dye. d Conjugation of azide-modified Cy5 with TPT3CO-modified RNA via Click chemistry.

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