Real-time measurement of in vitro transcription.

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
JournalNucleic acids research
Volume32
Issue number9
DOIs
StatePublished - Jan 1 2004

Fingerprint

DNA-Directed RNA Polymerases
Fluorescence
RNA
Deoxyribonucleotides
Fluorescent Dyes
In Vitro Techniques

All Science Journal Classification (ASJC) codes

  • Genetics

Cite this

@article{8d3da11b1422428d908243117ed0b55f,
title = "Real-time measurement of in vitro transcription.",
abstract = "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.",
author = "Marras, {Salvatore A.E.} and Benjamin Gold and Kramer, {Fred Russell} and Issar Smith and Sanjay Tyagi",
year = "2004",
month = "1",
day = "1",
doi = "10.1093/nar/gnh068",
language = "English (US)",
volume = "32",
journal = "Nucleic Acids Research",
issn = "0305-1048",
publisher = "Oxford University Press",
number = "9",

}

Real-time measurement of in vitro transcription. / Marras, Salvatore A.E.; Gold, Benjamin; Kramer, Fred Russell; Smith, Issar; Tyagi, Sanjay.

In: Nucleic acids research, Vol. 32, No. 9, 01.01.2004.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Real-time measurement of in vitro transcription.

AU - Marras, Salvatore A.E.

AU - Gold, Benjamin

AU - Kramer, Fred Russell

AU - Smith, Issar

AU - Tyagi, Sanjay

PY - 2004/1/1

Y1 - 2004/1/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=2442699226&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=2442699226&partnerID=8YFLogxK

U2 - 10.1093/nar/gnh068

DO - 10.1093/nar/gnh068

M3 - Article

C2 - 15155820

AN - SCOPUS:2442699226

VL - 32

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 9

ER -