Enzymatic Ligation Strategies for the Preparation of Purine Riboswitches with Site-Specific Chemical Modifications

Abstract

One of the most versatile riboswitch classes refers to purine nucleoside metabolism. In the cell, purine riboswitches of the respective mRNAs either act at the transcriptional or translational level and off- or on-regulate genes upon binding to their dedicated ligands. Biophysical studies on ligand-induced folding of these RNA domains in vitro contribute to understanding their regulation mechanisms in vivo. For such studies, in particular, for approaches using fluorescence spectroscopy, the preparation of large RNAs with site-specific chemical modifications is required. Here, we describe a strategy for the preparation of riboswitch aptamers and aptamers adjoined to their expression platforms by chemical synthesis and enzymatic ligation. The modular design enables fast access to a large number of purine riboswitch derivatives with the modification of interest at any strand position. We exemplarily provide a detailed protocol for the preparation of adenosine deaminase (add) A-riboswitch variants with 2-aminopurine (AP) modifications at the 40-nmol scale.

Probing RNA dynamics via longitudinal exchange and CPMG relaxation dispersion NMR spectroscopy using a sensitive 13C-methyl label

“…For even larger molecules, the signal-to-noise limitation could be alleviated by using cryogenic probes along with TROSY variants of the NMR experiments (17,46). The labeling procedure itself is feasible for RNAs of up to 150 nt using the recently developed approach that combines chemical RNA synthesis and enzymatic ligation methods (47,48). …”

NSUN 3 and ABH 1 modify the wobble position of mt‐t RNA Met to expand codon recognition in mitochondrial translation

“…Synthetic tRNAs were prepared by enzymatic ligation of chemically synthesised RNA fragments using T4 DNA ligase and DNA splint oligonucleotides (2–5 nmol scale, incubation at 30°C for 12 h), analogous to previously reported procedures (Rieder et al , 2009). The full‐length tRNAs were isolated by denaturing PAGE, extracted into Tris–NaCl buffer, precipitated with ethanol and re‐dissolved in water.…”

“…RNA oligonucleotides were prepared by solid-phase synthesis using 2 0 -O-TOM-protected ribonucleotide phosphoramidites, chemically phosphorylated on solid support, deprotected in two steps with methylamine in water/ethanol, followed by 1 M tetrabutylammonium fluoride in tetrahydrofuran, purified by denaturing PAGE, and analysed by analytical anion exchange chromatography under (Rieder et al, 2009). The full-length tRNAs were isolated by denaturing PAGE, extracted into Tris-NaCl buffer, precipitated with ethanol and re-dissolved in water.…”

NSUN6 is a human RNA methyltransferase that catalyzes formation of m5C72 in specific tRNAs

“…RNA oligonucleotides were prepared by solid-phase synthesis using 2 ′ -O-TOM-protected ribonucleotide phosphoramidites, (ChemGenes) chemically phosphorylated on the solid support, deprotected in two steps with methylamine in water/ethanol, followed by 1 M tetrabutylammonium fluoride in tetrahydrofuran, purified by denaturing PAGE, and analyzed by analytical anion exchange chromatography and ESI-MS. Synthetic tRNAs were prepared by enzymatic ligation of chemically synthesized RNA fragments using T4 DNA ligase (Fermentas) and DNA splint oligonucleotides (2-5 nmol scale, incubation at 30°C for 12 h), analogous to previously reported procedures (Rieder et al 2009). The full-length tRNAs were isolated by denaturing PAGE, extracted into Tris-NaCl buffer, precipitated with ethanol and re-dissolved in water.…”