Site-specific dual-color labeling of long RNAs for single-molecule spectroscopy

Abstract

Labeling of long RNA molecules in a site-specific yet generally applicable manner is integral to many spectroscopic applications. Here we present a novel covalent labeling approach that is site-specific and scalable to long intricately folded RNAs. In this approach, a custom-designed DNA strand that hybridizes to the RNA guides a reactive group to target a preselected adenine residue. The functionalized nucleotide along with the concomitantly oxidized 3′-terminus can subsequently be conjugated to two different fluorophores via bio-orthogonal chemistry. We validate this modular labeling platform using a regulatory RNA of 275 nucleotides, the btuB riboswitch of Escherichia coli, demonstrate its general applicability by modifying a base within a duplex, and show its site-selectivity in targeting a pair of adjacent adenines. Native folding and function of the RNA is confirmed on the single-molecule level by using FRET as a sensor to visualize and characterize the conformational equilibrium of the riboswitch upon binding of its cofactor adenosylcobalamin. The presented labeling strategy overcomes size and site constraints that have hampered routine production of labeled RNA that are beyond 200 nt in length.

Site-Specific Fluorescent Labeling of RNA Interior Positions

“…Zhao et al [ 68 , 69 ] have developed a novel approach to specifically transfer an alkyne group to a specific adenosine residue, even one present in a double-stranded region, in a four step sequence. As illustrated in Figure 6 , complementary DNA Helper sequences first unfold secondary structure neighboring the targeted A (step i).…”

“…Of note, the periodate oxidation step iii also oxidizes the RNA 3′-terminus to a dialdehyde, permitting its coupling with a second fluor by an orthogonal chemistry. This double labeling approach was used to generate FRET signals from labeled A residues targeted in both single-stranded [ 71 ] and double-stranded regions [ 68 , 69 ].…”

Encapsulation of Fluorescently Labeled RNAs into Surface-Tethered Vesicles for Single-Molecule FRET Studies in TIRF Microscopy

“…Site-specific labelling can also be achieved by DNA-helper-strand-guided post-transcriptional base modification and subsequent bi-orthogonal RNA labelling as presented recently [32].…”

Ensemble and single-molecule FRET studies of protein synthesis

“…In addition to splinted ligation, labeling at specific internal positions of in vitro transcribed RNA can be mediated by a complementary DNA oligonucleotide containing a reactive group at either the 5’ or 3’ ends [67, 68]. However, these oligonucleotide-guided mRNA labeling strategies have not yet been widely used in FRET studies.…”