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 language | English (US) |
|---|---|
| Journal | Nucleic acids research |
| Volume | 32 |
| Issue number | 9 |
| DOIs | |
| State | Published - Jan 1 2004 |
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All Science Journal Classification (ASJC) codes
- Genetics
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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 journal › Article
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 -