Real-time measurement of in vitro transcription.
We have developed a simple method to measure RNA synthesis in real time. In this technique, transcription reactions are performed in the presence of molecular beacons that possess a 2'-O-methylribonucleotide backbone. These probes become fluorescent as they hybridize to nascent RNA during the course of synthesis. We found that molecular beacons synthesized from natural deoxyribonucleotides were not suitable, because they are copied by RNA polymerases, generating complementary product strands that bind to the molecular beacons, causing a conformational change that results in unwanted fluorescence. However, when the molecular beacons are synthesized from 2'-O-methylribonucleotides, they are not copied and fluorescence is strictly dependent upon transcription of the added template. Utilizing these modified molecular beacons, quantitative comparisons were made of the activity of a variety of RNA polymerases and the effect of an inhibitor of transcription was determined.
Marras
Salvatore A E
SA
Public Health Research Institute, 225 Warren Street, Newark, NJ 07103, USA. marras@phri.org
Gold
Benjamin
B
Kramer
Fred Russell
FR
Smith
Issar
I
Tyagi
Sanjay
S
D017123:Bacteriophage T7 / Q000201:enzymology; D020029:Base Pairing; D001483:Base Sequence; D002474:Cell-Free System; D004247:DNA / Q000235:genetics / Q000378:metabolism; D015342:DNA Probes / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors / Q000378:metabolism; D005453:Fluorescence; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D012265:Ribonucleotides / Q000235:genetics / Q000378:metabolism; D012293:Rifampin / Q000494:pharmacology; D013698:Templates, Genetic; D013997:Time Factors; D014158:Transcription, Genetic
D015342:DNA Probes; D015345:Oligonucleotide Probes; D012265:Ribonucleotides; D012313:RNA; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin