Related Experiment Videos
Oligoribonucleotide-based gene-specific transcription inhibitors that target the open complex
L Milne1, D M Perrin, D S Sigman
1Department of Biological Chemistry, School of Medicine, and Molecular Biology Institute, UCLA, Los Angeles, California 90095-1570, USA.
Abstract:
We have demonstrated that oligoribonucleotides that lack a 3'-OH group and cannot be extended by RNA polymerase can hybridize to the single-stranded DNA formed inside the transcription initiation bubble (or open complex) and inhibit transcription. Using the lacUV5/Escherichia coli RNA polymerase or trpEDCBA/E. coli RNA polymerase transcription system as a model, we have found that effective inhibitors are five nucleotides in length and must be complementary to the DNA template strand in the region from -5 to +2 about the transcription start site (designated +1). We have used the DNA cleavage activity of 1,10-phenanthroline-copper to confirm that the mechanism of inhibition is via oligoribonucleotide hybridization to the open complex and have used this cleavage chemistry to demonstrate that these oligonucleotide inhibitors hybridize in an antiparallel orientation to their DNA target. Systematic modification of the parent phosphodiester oligoribonucleotide pentamer revealed that the phosphorothioate backbone-containing analogs have increased open complex binding affinity and are more effective transcription inhibitors than their phosphodiester counterparts.
Insights
Modified oligoribonucleotides that cannot be extended by RNA polymerase inhibit transcription by binding to the DNA open complex. Phosphorothioate analogs show enhanced binding and inhibition compared to standard oligoribonucleotides.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Transcription initiation involves the formation of an open complex, a single-stranded DNA bubble.
- Oligonucleotides can potentially interfere with biological processes by binding to nucleic acids.
Purpose of the Study:
- To investigate the potential of modified oligoribonucleotides as inhibitors of transcription.
- To elucidate the mechanism of inhibition and identify optimal inhibitor characteristics.
Main Methods:
- Utilized lacUV5/Escherichia coli RNA polymerase and trpEDCBA/E. coli RNA polymerase transcription systems.
- Employed 1,10-phenanthroline-copper to probe oligonucleotide hybridization and DNA binding.
- Synthesized and tested phosphodiester and phosphorothioate oligoribonucleotide analogs.
Main Results:
- Oligoribonucleotides lacking a 3'-OH group inhibit transcription by hybridizing to the DNA template strand within the open complex.
- Effective inhibitors are pentamers complementary to the DNA template from -5 to +2 relative to the transcription start site.
- Phosphorothioate analogs exhibit increased binding affinity and enhanced transcription inhibition compared to phosphodiester counterparts.
- Oligonucleotide hybridization occurs in an antiparallel orientation to the DNA target.
Conclusions:
- Modified oligoribonucleotides represent a viable strategy for inhibiting transcription.
- The phosphorothioate backbone enhances the efficacy of these transcription inhibitors.
- Understanding the precise binding interactions within the open complex is crucial for designing effective inhibitors.