Site‐Specific Fluorescent and Affinity Labelling of RNA by Using a Small Engineered Twin Ribozyme

Abstract

Over the past decade, RNA has become a focus of investigation into structure-function relationships. A large number of methods for structural studies of RNA are available. Application of those techniques often requires decoration of the sample with reporter groups and modifications such as fluorophores, cross-linking reagents, phosphorothioates, affinity tags or ESR spin labels, most desirably at a specific position.[1] We have developed a strategy for RNA modification that relies on a small engineered twin ribozyme that mediates the exchange of a patch of residing sequence of substrate RNA with a separately added synthetic RNA fragment.[2] Here we show that RNA fragments conjugated with fluorescent dyes or biotin are well accepted for strand exchange. Up to 53 % of a dye-labelled oligoribonucleotide has been inserted into a 145-mer RNA. Thus, for the first time, specific labelling of a long transcribed RNA at an internal predetermined position is demonstrated.Modified nucleosides can be site-specifically incorporated into RNA by chemical synthesis with modified nucleoside phosphoramidites. While this is a useful strategy for modification of synthetically available RNAs, modification of long transcripts or natural RNA requires alternative techniques. In this case, specific labelling is possible at the two ends by taking advantage of the unique reactivity of the RNA termini.[3] Functionalization at internal sites can be achieved by adding modified nucleoside triphosphates to the transcription mixture. [4] However, the range of modified nucleosides that can be incorporated during transcription is limited by the specificity of the polymerase, and the label becomes distributed over the entire molecule. The recently published procedure of indirect labelling through oligonucleotide hybridization is a useful alternative. However, it is restricted by the availability of specific hybridization sites in the folded state of the molecule.[5]We have developed a procedure for manipulating at will a chosen patch of a given RNA sequence. A small engineered twin ribozyme promotes, in a strictly controlled fashion, two RNA-cleavage events and two ligations, and thus mediates the specific exchange of RNA patches.[2] The strategy relies on the cleavage/ligation characteristics of the hairpin ribozyme, [6] a small naturally occurring catalytic RNA. Twin ribozymes are derived from tandem duplication of the hairpin ribozyme and thus inherit cleavage as well as ligation activity. Efficient fragment exchange is achieved by destabilization of the duplex between the ribozyme and the RNA patch to be removed (dark grey sequence (lower case letters) in Scheme 1 a) and stabilization of the duplex between ribozyme and fragment to be inserted (light grey sequence). After cleavage, the sequence patch (in lower case letters) is readily released from the ribozyme-substrate complex due to the GAUU tetraloop designed to weaken its binding. The new fragment (light grey) contains the four additional nucleotides complementary to the GAUU tetraloop and ...

Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry

“…Besides the chemical modification of the 3′-terminal vicinal diol (14,15), various enzymatic methods employing ligases (16–18) or nucleotidyl transferases, like terminal deoxynucleotidyl transferase (TdT) (19) or poly(A) polymerase (PAP) (20) have been reported. For post-synthetic, internal labeling of RNA, DNAzymes (21), twin ribozymes (22) or hybridization-based functionality transfer reactions (23) can be used, all of which rely on chemically synthesized, modified oligonucleotides. Although the conversion of terminal modifications to internal ones by ligases or nucleotidyl transferases has been reported (7), this approach is limited to the introduction of small modifications.…”

A two-stage mechanism of viral RNA compaction revealed by single molecule fluorescence

“…Many strategies have been developed for covalent labeling of proteins, ranging from relatively non-specific lysine-reactive dye conjugates to more selective cysteine-reactive maleimide derivatives and CLICK chemistry 62 - 66 . Specific covalent labeling of RNA can be achieved at either 5′ or 3′ termini 67 - 71 . Two 3′-end strategies were employed in the work described here: (1) end ligation of a dye labeled dA 10 oligomer; (2) incorporation of 3′-amino adenosine by poly-A-polymerase followed by amine-specific labeling 4 .…”

Sequence-controlled RNA self-processing: computational design, biochemical analysis, and visualization by AFM

“…Hairpin ribozymes that form a stable structure, such that fragments remain bound, favor ligation, whereas hairpin ribozymes that are less stable, such that cleavage fragments can easily dissociate, favor cleavage (Fedor 1999, Welz et al 2003. These characteristic features distinguish the hairpin ribozyme from other small ribozymes, and we have shown in previous work that structural manipulation of hairpin ribozyme variants allows tuning of cleavage and ligation activity (Welz et al 2003;Ivanov et al 2005;Vauleon et al 2005;Drude et al 2007Drude et al , 2011Pieper et al 2007;Müller 2013, Balke et al 2014). Among these variants is a hairpin ribozyme that can cleave off its 5 ′ -and 3 ′ -end (Pieper et al 2007).…”