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 gels of Pluronic F127 was used to analyze unfractionated in vitro transcription reactions and anion-exchange high-performance liquid chromatography (HPLC) fractions from transcription reactions. Guanosine monophosphate (GMP), the four nucleoside triphosphates (NTPs), abortive transcripts, and transcripts with lengths near the desired product length were simultaneously resolved and quantified in a single run. Oligonucleotides up to at least 35 nucleotides 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 20% Pluronic F127 at pH 8.3 in Tris-borate-EDTA (TBE) buffer (30 degrees C). 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 little decrease in performance, and for up to 100 runs with performance acceptable for many applications. Higher electric fields appeared to affect the gel structure and necessitated more frequent capillary refilling. Capillary gel electrophoresis (CGE) of HPLC fractions in Pluronic gels facilitates rapid recovery of RNA products and the large remaining amounts of valuable, isotopically labeled NTPs. In addition, comparison of electrophoretic patterns under denaturing and nondenaturing conditions yields insights into potential conformational heterogeneity of the folded nucleic acid states. CGE in Pluronic gels provides a rapid, highly discriminating means for analyzing in vitro transcription reactions.
Epperson
J D
JD
Department of Chemistry and Institute of Molecular Biophysics, The Florida State University, Tallahassee 32306-4390, USA.
Dodge
J
J
Rill
R L
RL
Greenbaum
N L
NL
D002474:Cell-Free System; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D019075:Electrophoresis, Capillary / Q000379:methods*; D005782:Gels; D009711:Nucleotides / Q000032:analysis*; D009843:Oligoribonucleotides / Q000032:analysis*; D020442:Poloxamer / Q000737:chemistry*; D012313:RNA / Q000032:analysis* / Q000096:biosynthesis / Q000302:isolation & purification; D014158:Transcription, Genetic
D005782:Gels; D009711:Nucleotides; D009843:Oligoribonucleotides; D020442:Poloxamer; D012313:RNA