Gel electrophoresis in a polyvinylalcohol coated fused silica capillary for purity assessment of modified and secondary-structured oligo- and polyribonucleotides

Abstract

Application of a polyvinylalcohol-coated (PVA-coated) capillary in capillary gel electrophoresis (CGE) enables the selective separation of oligoribonucleotides and their modifications at high resolution. Quality assessment of shorter oligomers of small interfering RNA (siRNA) is of key importance for ribonucleic acid (RNA) technology which is increasingly being applied in medical applications. CGE is a technique of choice for calculation of chemically synthesized RNAs and their modifications which are frequently obtained as a mixture including shorter oligoribonucleotides. The use of CGE with a PVA-coated capillary to analyze siRNA mixtures presents an alternative to conventionally employed techniques. Here, we present study on identification of the length and purity of RNA mixture ingredients by using PVA-coated capillaries. Also, we demonstrate the use of PVA-coated capillaries to identify and separate phosphorylated siRNAs and secondary structures (e.g. siRNA duplexes).

HPLC methods for purity evaluation of man-made single-stranded RNAs

“…Excellent resolution with up to 60nt long RNA oligos was reported recently using CGE with a polyvinylalcohol coated fused silica capillary 33 . It is likely that this method could be extended to include longer RNAs, and yield a strictly size-dependent separation, well documented for CGE.…”

“…Other modes, like size-exclusion, affinity, and hydrophobic interaction have also been used in special cases. State-of-the-art in RNA oligo separations is currently up to the 60nt using capillary gel electrophoresis (CGE) 33 , or HPLC in conjunction with IP-RP 18,34–36 or with IEX modes 37,38 . Limited number of companies and research groups 17 claim synthesis, analysis, identification, and purification of RNA oligos at the 100nt level.…”

Comparison of capillary electrophoresis and zwitterionic-hydrophilic interaction capillary liquid chromatography with ultraviolet and mass spectrometry detection for the analysis of microRNA biomarkers

“…As miRNA are oligonucleotides, they are very polar compounds with ionizable phosphate groups. Therefore, capillary electrophoresis (CE) [12][13][14][15][16][17] and hydrophilic interaction capillary liquid chromatography (capHILIC) [18,19] can be regarded nowadays as very suitable microscale separation techniques for their highly efficient separation and direct detection. Furthermore, under certain conditions these techniques are compatible with mass spectrometry (MS) detection [16][17][18][19], which allows identifying the separated miRNAs and characterizing their post-transcriptional modifications.…”

“…Capillary gel electrophoresis has been extensively applied to separate oligonucleotides, including miRNAs [12][13][14][15]20], but the typical sieving conditions are non-compatible with on-line MS detection. Separation conditions in capillary zone electrophoresis, hereafter referred to as CE, can be optimized for capillary electrophoresis-mass spectrometry (CE-MS).…”

Voltage‐programming‐based capillary gel electrophoresis for the fast detection of angiotensin‐converting enzyme insertion/deletion polymorphism with high sensitivity

“…However, the SGE methods need large sample injection amounts and a time consuming processes with low resolution. By contrast, CGE has become an alternative method for separating DNA fragments due to its fast speed, high resolution, and low reagent consumptions .…”