Site-Selective RNA Functionalization via DNA-Induced Structure

Abstract

Methods for RNA functionalization at specific sites are in high demand but remain a challenge, particularly for RNAs produced by transcription rather than by total synthesis. Recent studies have described acylimidazole reagents that react in high yields at 2'-OH groups stochastically at nonbase-paired regions, covering much of the RNA in scattered acyl esters. Localized reactions, if possible, could prove useful in many applications, providing functional handles at specific sites and sequences of the biopolymer. Here, we describe a DNA-directed strategy for in vitro functionalization of RNA at site-localized 2'-OH groups. The method, RNA Acylation at Induced Loops (RAIL), utilizes complementary helper DNA oligonucleotides that expose gaps or loops at selected positions while protecting the remainder in DNA-RNA duplexes. Reaction with an acylimidazole reagent is then carried out, providing high yields of 2'-OH conjugation at predetermined sites. Experiments reveal optimal helper oligodeoxynucleotide designs and conditions for the reaction, and tests of the approach are carried out to control localized ribozyme activities and to label RNAs with dual-color fluorescent dyes. The RAIL approach offers a simple and novel strategy for site-selective labeling and control of RNAs, potentially of any length and origin.

Figure 1.

Schematic of the RAIL approach for site-selective RNA acylation. The RNA of interest (R) is incubated with one or more complementary helper DNAs (H) to protect most of the RNA, but leaving a single unpaired nucleotide in a loop (a) or gap (b). The exposed 2′-OH group can then selectively react with an acylating reagent, such as the prototypical NAI-N₃, yielding a site-defined functionalized RNA after removing the helper DNA. Note that loops and gaps larger than a single nucleotide are also possible.

Figure 2.

Optimizing helper DNA protection from random RNA acylation. (a) Schematic of acylation of single stranded RNA (ssRNA) and RNA-DNA duplex with a full DNA complement. (b) MALDI-TOF mass spectrum of acylated ssRNA (R), RNA protected by fully complementary DNA (R/H), RNA protected by fully complementary DNA with three deoxyadenosines overhanging at both ends (R/H_o) and 2′-deoxy-3′-phosphate modified RNA protected by fully complementary DNA with 3a overhang (R_p/H_o); reacting with 50 mM NAI-N3 at 37°C for 4h in MOPS buffer. (c) Gel electrophoretic analysis of reverse transcriptase (RT) primer extension reveals a general lack of stops due to protected RNA, save for trace reaction at a central C, U site.

Figure 3.

Characterization of RNA acylation at DNA-induced loops. (a) Schematic showing the process of induced loop RNA acylation. (b) MALDI-TOF mass spectra of DNA-protected RNA (R_p/H_o), DNA-induced 1nt bulge RNA (R_p/H_o-L1), and DNA-induced 3nt loop RNA (R_p/H_o-L3), reacting with 200 mM NAI-N₃. (c) Gel electrophoretic analysis of RT stops for the RNA samples with no loop (R/H_o), 1nt bulge (R/H_o-L1) and 3nt loop (R/H_o-L3) reacting with 200 mM NAI-N₃. The sequence and sites of the bands in the gel are as shown. Note that RT stops (and corresponding bands) occur at the nucleotide immediately 3′ to the acylated residue.

Figure 4.

Characterization of RNA acylation at induced gaps. (a) Schematic for the steps of induced-gap RNA acylation. (b) Gel electrophoretic analysis of RT stops for RNA samples with no gap (R/H_o), nick (R/H_o-N), 1nt gap (R/H_o-G1) and 3nt gap (R/H_o-G3), reacting with 200 mM NAI-N₃. Sequences and sites are as indicated.

Figure 5.

Programming local acylation sites by shifting positions of loops or gaps in a 39mer RNA target. PAGE analysis of RT-stops for the samples with 1nt bulges or gaps at different positions as shown. The aligned sequences of the shifting bands in the gel are as indicated. Note that RT stops (and corresponding bands) occur at the nucleotide immediately 3′ to the reactive position.

Figure 6.

RAIL approach for the site-selective programmable control of a tandem ribozyme. (a) Sequences of a tandem ribozyme (TR) with two catalytic cores (3TR, 5TR) and two dually labeled substrates of 3S for 3TR, 5S for 5TR; Selectively acylation of TR was shown on the below via RAIL method, resulting in a 3TR acylated ribozyme and a 5TR acylated ribozyme. (b) Mechanisms of RAIL method enabled site-specific control of the tandem ribozyme. (c) Plot showing the relative initial rates of each substrate (3S and 5S) cleavage relative to that of unreacted TR (normalized to 1.0). Error bars represent the standard deviation of triplicate experiments.

Figure 7.

Successive RAIL approach for the dual labeling of a 65nt small nucleolar RNA (SNORD78) and observation of folding by FRET. (a) SNORD78 RNA sequence with labeling sites (G14 with Alex488; A49 with TAMRA) marked in blue. Schematic for the process of RNA dual labeling via two successive reactions. (b) Image of gel analysis of double-labeled RNA; overlay of Alex488 (green) and TAMRA (red) channels showing coincidence of the two dyes. (c) FRET signals respond to changing RNA structure in buffer versus water (fluorescence emission on donor excitation at 490 nm).