Analysis of oligonucleotides and unincorporated nucleotides from in vitro transcription by capillary electrophoresis in Pluronic F127 gels

Small functional RNAs required for structure studies are often prepared by in vitro transcription. Capillary electrophoresis in liquid crystalline gel s of Pluronic F127 was used to analyze unfractionated in vitro transcriptio n reactions and anion-exchange high-performance liquid chromatography (HPLC ) fractions from transcription reactions. Guanosine monophosphate (GMP), th e four nucleoside triphosphates (NTPs), abortive transcripts, and transcrip ts with lengths near the desired product length were simultaneously resolve d and quantified in a single run. Oligonucleotides up to at least 35 nucleo tides were resolved to baseline within 10 min using a moderate field (185 V /cm) and short effective capillary length (7.6 cm) for electrophoresis in 2 0% Pluronic F127 at pH 8.3 in Tris-borate-EDTA (TBE) buffer (30 degreesC). Nucleotide migration times were 4-5 min, in the order UTP+CTP (unresolved) <ATP<GTP<GMP. A single capillary filling was reused for 40-50 runs with lit tle decrease in performance, and for up to 100 runs with performance accept able for many applications. Higher electric fields appeared to affect the g el structure and necessitated more frequent capillary refilling. Capillary gel electrophoresis (CGE) of HPLC fractions in Pluronic gels facilitates ra pid recovery of RNA products and the large remaining amounts of valuable, i sotopically labeled NTPs. In addition, comparison of electrophoretic patter ns under denaturing and nondenaturing conditions yields insights into poten tial conformational heterogeneity of the folded nucleic acid states. CGE in Pluronic gels provides a rapid, highly discriminating means for analyzing in vitro transcription reactions.