Related Experiment Videos
Mechanisms of the inhibition of reverse transcription by antisense oligonucleotides
C Boiziau1, N T Thuong, J J Toulmé
1Laboratoire de Biophysique Moléculaire, Université de Bordeaux II, Institut National de la Santé et de la Recherche Médicale CJF 90-13, France.
Abstract:
We have demonstrated that the synthesis of cDNA by avian myeloblastosis virus and Moloney murine leukemia virus reverse transcriptases can be prevented by oligonucleotides bound to the RNA template approximately 100 nucleotides remote from the 3' end of the primer. The RNA was truncated at the level of the antisense oligonucleotide-RNA duplex during the reverse transcription. The key role played by the reverse transcriptase-associated RNase H activity in the inhibition process was shown by the use of (i) inhibitors of RNase H (NaF or dAMP), (ii) Moloney murine leukemia virus reverse transcriptase devoid of RNase H activity, or (iii) alpha-analogues of oligomers that do not elicit RNase H-catalyzed RNA degradation. In all three cases the inhibitory effect was either reduced (NaF, dAMP) or totally abolished. However, an alpha-oligomer bound to the sequence immediately adjacent to the primer-binding site prevented reverse transcription. Therefore, initiation of polymerization can be blocked by means of an RNase H-independent mechanism, whereas arrest of a growing cDNA strand can be achieved only by an oligonucleotide mediating cleavage of the template RNA.
Insights
Oligonucleotides can inhibit reverse transcriptase activity by targeting RNA templates. RNase H activity is crucial for blocking growing cDNA strands, while initiation can be blocked independently.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Reverse transcriptases are crucial enzymes in retroviral replication.
- Oligonucleotides can be designed to interact with RNA templates.
- RNase H activity is associated with some reverse transcriptases and degrades RNA.
Purpose of the Study:
- To investigate the mechanism by which oligonucleotides inhibit reverse transcriptase activity.
- To determine the role of RNase H in oligonucleotide-mediated inhibition.
- To explore strategies for blocking cDNA synthesis.
Main Methods:
- Using avian myeloblastosis virus and Moloney murine leukemia virus reverse transcriptases.
- Employing antisense oligonucleotides bound to RNA templates.
- Utilizing RNase H inhibitors (NaF, dAMP) and RNase H-deficient reverse transcriptase.
- Testing alpha-analogues of oligomers.
Main Results:
- Oligonucleotides ~100 nucleotides from the primer binding site inhibited reverse transcription by truncating the RNA template.
- Inhibition was significantly reduced or abolished when RNase H activity was inhibited or absent.
- An alpha-oligomer adjacent to the primer-binding site blocked initiation independently of RNase H.
- Arresting cDNA synthesis required template RNA cleavage mediated by oligonucleotides.
Conclusions:
- Oligonucleotide-mediated inhibition of reverse transcriptase is dependent on RNase H activity for arresting cDNA synthesis.
- RNase H-independent mechanisms can block the initiation of polymerization.
- Oligonucleotides targeting RNA template cleavage are effective in preventing reverse transcription.