Catalytic nucleic acid-based RNA labeling

Self-alkylating fluorescein-iodoacetamide reactive ribozyme selected by Sharma et al, is an example of a catalytic RNA that was directly selected for RNA labeling. The selection was performed using fluorescein-iodoacetamide and IP-SELEX (Figure 1-7 B) as mentioned in 1.3.2.4. The outcome of this selection were two ribozymes denoted as 1FR1 and 5FR1. These

ribozymes were able to self-alkylate at an undetermined internal position, in the presence of fluorescein-iodoacetamide. The fusion of these two ribozymes to a transcript coding for a part of the mCherry protein resulted in the labeling of the fusion construct in vitro and in cell lysate (Figure 1-16 A) (Sharma et al., 2014). The high specificity of these ribozymes towards Fluorescein-IA limits their application to single-color and single-probe labeling reactions.

Another example of self-alkylating ribozymes with the potential for RNA labeling is a 42-nucleotide long RNA catalyst derived from Aeropyrum pernix genomic RNA sequences (McDonald et al., 2014). To identify this ribozyme, McDonald et al. used a mixture of eight biotinylated compounds with electrophilic functionalities, in combination with an RNA pool of transcriptomic fragments derived from 9 different species. The self-alkylating ribozyme was isolated within 6 rounds of selection. Further characterization on this ribozyme revealed its specific reactivity towards di-substituted cis-epoxides (Figure 1-5 B). The self-alkylation was still observed even when the biotin was replaced by other moieties containing alkynyl or azido functionalities or even fluorescent dyes such as TAMRA. 5S rRNA and ASH1 mRNA were transcriptionally fused to this catalyst and were shown to be successfully labeled as pure transcript or in the context of total cellular RNA (Figure 1-16 B) (McDonald et al., 2014).

Figure 1-16 Cis-reacting self-alkylating ribozymes as RNA labeling tools. (A) Fluorescein iodoacetamide reactive ribozyme can be inserted into target RNA, allowing fluorescein labeling at internal positions. (B) Insertion of the epoxide reactive ribozyme into the ROI allows labeling using various epoxide containing chemical probes.

The Twin-ribozyme is an engineered form of the hairpin ribozyme, which has also been applied to RNA labeling in trans. As mentioned in 1.2, the hairpin ribozyme is a self-cleaving ribozyme. The cleavage reaction generates a 2'-3' cyclic phosphodiester and a 5'-OH at the ends of the RNA fragments, which can be rejoined, since the hairpin ribozyme can also catalyze the re-ligation reaction (Hampel and Tritz, 1989). The ribozyme can be engineered into a trans-acting variant consisting of a conserved catalytic core and a pair of binding arms.

The arms can be used to target the ribozyme to RNA sequences of interest (Walter and Burker, 1998). This engineered variant can catalyze a similar cleavage/re-ligation reaction on an independent RNA molecule in-trans. The twin-ribozyme was developed by tandem-connection of two copies of the hairpin ribozyme via a linker sequence (Schmidt et al., 2000;

Welz et al., 2003). The arms and the linker are designed complementary to the target RNA.

Upon hybridization, twin-ribozyme facilitates excision of an RNA segment and its replacement with a synthetic piece carrying a label at a desired position. For re-ligation to occur, the ligated patch needs to carry a 2'-3'cyclic phosphodiester at its 3'-end. Since the original piece can also be ligated back, the linker is designed to form an unpaired tetraloop when bound to target RNA. The synthetic patch, however, is made fully complementary to the linker sequence. This is to ensure that the synthetic piece can replace the excised fragment more efficiently (Figure 1-17) (Vauléon et al., 2005).

Figure 1-17 Twin-ribozyme based RNA labeling. Twin-ribozyme labels target RNA via strand exchange mechanism. A synthetic RNA piece carrying a variety of labels (red)can be used to replace a target segment (cyan). 3'-cyclic phosphodiester (cp) is essential to the targeting reaction. The tetraloop region (green) remains unpaired to the original segment while pairing to the synthetic piece to allow efficient strand exchange.

Samanta et al, report another RNA labeling method using a polymerase ribozyme (Samanta et al., 2018). Polymerase ribozymes are a class of artificial RNA catalysts that can catalyze template-based extension of DNA or RNA primers using NTPs (McGinness and Joyce, 2003). Samanta et al, exploited polymerase ribozyme 24-3, for template-dependent addition of labeled nucleotides to 3'-end of RNA or DNA (Figure 1-18). The RNA of interest in this system acts as a primer annealed to a template oligonucleotide. The template oligonucleotide and the ribozyme are designed complementary at their 5'-end (Figure 1-18). The segment of the ribozyme complementary to the template is called tag sequence. This complementarity facilitates primer-template pair recognition by the polymerase ribozyme. The template is designed in a manner that only a single nucleotide extension to the RNA of interest is permissible. The NTP analogs added using this strategy could bear a diverse set of

functionalities and modifications at various positions. This system has only so far been used in vitro. Live-cell application of this ribozyme is challenging due to the lack of orthogonality towards natural NTPs (Samanta et al., 2018). the application of this ribozyme is also only limited to the 3'-end labeling. This may cause additional complications particularly when it comes to specific targeting of 3'-polyadenylated RNA.

Figure 1-18 24-3 polymerase ribozyme mediated RNA labeling. Tag sequence (green) at 5'-end of the ribozyme is designed complementary to the 5'-end of the template. The ROI in this method acts as a primer. Annealing of ROI to the template allows one nucleotide extension in the presence of the appropriate NTP analog. The NTP analog can carry a variety of labels at various positions.