Lee R, Kaushik N, Modak M J, Vinayak R, Pandey V N
Department of Biochemistry and Molecular Biology, UMD-New Jersey Medical School, Newark 07103, USA.
Biochemistry. 1998 Jan 20;37(3):900-10. doi: 10.1021/bi972197m.
We report here that polyamide nucleic acid (PNA) as well as a polyamide nucleic acid-DNA chimera complementary to the primer binding site of the HIV-1 genome can completely block priming by tRNA3Lys and consequently the in vitro initiation of reverse transcription by HIV-1 RT. Conventional heating and cooling is not required for annealing PNA analogs to the complementary nucleotide sequence as effective blockage of reverse transcription results from their invasion in the duplex region of preprimed U5-PBS HIV-1 RNA template-primer and was seen even at ambient temperature. Further, the extension of the initiated nascent (-) strand DNA can also be blocked by inclusion of another PNA, targeted to upstream sequences in the U5 region of the viral RNA. Interestingly, a PNA chimera having only two DNA nucleotides annealed with the U5-PBS RNA is recognized as a bonafide primer by HIV-1 RT, as the 3'OH end of the chimeric molecule is extended by the enzyme in the presence of dNTPs. A significant observation was that RNA/PNA or RNA/(PNA-DNA) hybrids were entirely resistant to the RNase H activity of HIV-1 RT. Furthermore, PNA invasion into the RNA/DNA hybrid completely prevented the cleavage of the RNA strand, suggesting that the RNase H activity of HIV-1 RT which was required in reverse transcription may also be inhibited by the PNA oligomer. These observations suggest that oligomeric PNAs targeted to various critical regions of the viral genome are likely to have strong therapeutic potential for interrupting multiple steps involved in the replication of HIV-1 and warrant serious investigation especially in the area of an effective delivery system.
我们在此报告，聚酰胺核酸（PNA）以及与HIV-1基因组引物结合位点互补的聚酰胺核酸-DNA嵌合体可完全阻断tRNA3Lys引发的反应，从而阻断HIV-1逆转录酶在体外启动的逆转录过程。将PNA类似物与互补核苷酸序列退火不需要传统的加热和冷却，因为逆转录的有效阻断是由于它们侵入预引发的U5-PBS HIV-1 RNA模板-引物的双链区域，甚至在室温下也能观察到这种阻断效果。此外，通过加入另一种靶向病毒RNA U5区域上游序列的PNA，也可以阻断起始的新生（-）链DNA的延伸。有趣的是，仅含两个与U5-PBS RNA退火的DNA核苷酸的PNA嵌合体被HIV-1逆转录酶识别为真正的引物，因为在dNTP存在的情况下，该嵌合分子的3'OH末端会被该酶延伸。一个重要的观察结果是，RNA/PNA或RNA/（PNA-DNA）杂交体对HIV-1逆转录酶的RNase H活性完全具有抗性。此外，PNA侵入RNA/DNA杂交体完全阻止了RNA链的切割，这表明HIV-1逆转录酶在逆转录过程中所需的RNase H活性也可能被PNA寡聚物抑制。这些观察结果表明，靶向病毒基因组各个关键区域的寡聚PNA可能具有强大的治疗潜力，可中断HIV-1复制过程中的多个步骤，尤其在有效递送系统领域值得深入研究。
