Efficient Large-Scale Preparation and Purification of short Single-Stranded RNA Oligonucleotides

Abstract

Sequence-specific RNA recognition by RNA-binding proteins plays a crucial role in the post-translational regulation of gene expression. Biophysical and biochemical studies help to unravel the principles of sequence-specific RNA recognition, but the methods used require large amounts of single-stranded RNA (ssRNA). Here we present a fast and robust method for large-scale preparation and purification of short ssRNA oligonucleotides for biochemical, biophysical, and structural studies. We designed an efficiently folding, self-cleaving hammerhead (HH) ribozyme to prepare ssRNA oligonucleotides. Hammerhead ribozyme RNAs self-cleave with over 95% efficiency during in vitro transcription as a function of magnesium concentration to produce high yields of the desired ssRNA products. The resulting ssRNAs can be purified from crude transcription reactions by denaturing anion-exchange chromatography and then desalted by weak anion-exchange chromatography using volatile ammonium bicarbonate buffer solutions. The ssRNA oligonucleotides produced this way are homogenous, as judged by mass spectrometry (MS), and are suitable for biochemical and biophysical studies. Moreover, for high-resolution NMR structure determination of RNA-protein complexes, our protocol enables efficient preparation of ssRNA oligonucleotides with various isotope-labeling schemes which are not commercially available.

Recent Methods for Purification and Structure Determination of Oligonucleotides

“…The preparation of oligonucleotides is usually desalted, quantified, evaporated to dryness in a lyophilizer, and then supplied to the user in the form of power [13]. In this paper, we introduce the two main techniques, polyacrylamide gel electrophoresis (PAGE) and chromatography, which are widely used for the separation of target oligonucleotides [14,15].…”

hnRNP A1 induces aberrant CFTR exon 9 splicingviaa newly discovered Exonic Splicing Silencer element

“…The ssDNAs were purchased from Dharmacon. The 15-mer ssDNA (5'-CAGGGAT-TTGGGGAC-3'), the 7-mer ssDNAs (5'-CAGGGAT-3') and the three 8-mer ssDNAs (5'-TGGGGAAT-3', 5'-CAGGGATC-3', 5'-CTGGGCAC-3') were purified under denaturing conditions using the ultiMate 3000 HPLC system with an anion-exchange preparative DNAPac PA100 Nucleic Acid Column (both Thermo Fisher Scientific) as described before 23 . We performed the purification at 85°C with a flow rate of 20ml/min.…”

Applications of NMR to structure determination of RNAs large and small

“…The band corresponding to the RNA is excised from the gel and the RNA is extracted by electroelution, ethanol precipitation and extensive dialysis to refold the RNA and remove any leftover acrylamide. More recently, a set of chromatographic approaches has also been reported, which includes reverse-phase high-performance liquid chromatography (HPLC), anion-exchange HPLC, size exclusion chromatography and affinity based chromatography [11–14]. The chromatographic methods are faster, reduce sample losses and retain the RNA in its native conformation throughout, but it is not always possible to obtain sufficient purification of desired products from aborts.…”