Site-Specific Dual-Color Labeling of Long RNAs

Abstract

Labeling of large RNAs with reporting entities, e.g., fluorophores, has significant impact on RNA studies in vitro and in vivo. Here, we describe a minimally invasive RNA labeling method featuring nucleotide and position selectivity, which solves the long-standing challenge of how to achieve accurate site-specific labeling of large RNAs with a least possible influence on folding and/or function. We use a custom-designed reactive DNA strand to hybridize to the RNA and transfer the alkyne group onto the targeted adenine or cytosine. Simultaneously, the 3-terminus of RNA is converted to a dialdehyde moiety under the experimental condition applied. The incorporated functionalities at the internal and the 3-terminal sites can then be conjugated with reporting entities via bioorthogonal chemistry. This method is particularly valuable for, but not limited to, single-molecule fluorescence applications. We demonstrate the method on an RNA construct of 275 nucleotides, the btuB riboswitch of Escherichia coli.

Metal ions and sugar puckering balance single-molecule kinetic heterogeneity in RNA and DNA tertiary contacts

“…Making such a FRET construct would require multiple fragments to be ligated enzymatically. Recent advances in co-and post-transcriptional nucleic acid labeling now provide more flexibility in choosing dye positions, which are close to functionally important elements, and thus provide a basis for studying the kinetics of large RNA-protein assemblies on a single-molecule level 67,68 .…”