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Development of anti-virus agents on the basis of their ability to induce replicational errors

基于诱导复制错误的能力开发抗病毒剂

基本信息

批准号：
14572093
负责人：
NEGISHI Kazuo
金额：
$ 2.56万
依托单位：
OKAYAMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14572093/
关键词：
nucleotide analogs error catastrophe replicational error ambiguous base-pairing RNA virus retrovirus RNA polymerase HPLC PTP KTP エラーカタストロフィーポリオウイルス C型肝炎ウイルス supF 両義的な取組み

项目摘要

1. Synthesis of nucleotide analogs.We have established the methods for synthesis of N^4-hydroxycytidine triphosphate (N^4-OhCTP),N^4-methoxycytidine (N^4-MoCTP), and N^4-methycytidine (N^4-McCTP), and obtained pure materials. With these pure, samples, their incorporation into RNA catalyzed by T3 RNA polymerase was studied. RNA synthesis took place if N^4-OhCTP, or N^4-MoCTP was present in place of UTP, but not when they were added in place of CTP. This results shows that they behave only as UTP analogs in spite of their ambiguity in bass-pairing. In contrast, N^4-MeCTP behaved as CTP analog only. Incorporation properties of isoGTP, one of typical oxidation products from nucleotide, was studied using a commercial sample. It shows no incorporation by the RNA polymerase.2.Properties of the analogs in templates in the reversetranscription.Above finding enabled us to make RNA whose U or C was totally replaced with the analogs. Thus we have prepared RNA with analogs, and they were used as a template for reverse transcription with AMV reversetranscriptase. Resulting cDNA was amplified and analyzed with a DNA sequencer. The analysis indicated that N^4-hydroxycytidine was read as C and T almost equally. It suggests that this analog may cause mutation in a reversetranscription process.3. Anti-HIV virus activities of the analogs.Some analogs ware found to be very hopeful. Further study is continuing.4. Effects on RNA viruses and their polymerase.Action of analog triphosphates to Polio RNA polymerase was studied.5. Use of chitosan-triphosphate complexes.We have found that chitosan-triphosphate complex made by a reported method was not stable enough. Improvement of the protocol is currently underway.

1. 核苷酸类似物的合成。建立了N^4-羟基胞苷三磷酸（N^4-OhCTP）、N^4-甲氧基胞苷（N^4-MoCTP）和N^4-甲基胞苷（N^4-McCTP）的合成方法，并获得了纯材料。利用这些纯样品，研究了它们在T3 RNA聚合酶催化下与RNA的结合。如果用N^4-OhCTP或N^4-MoCTP代替UTP， RNA合成就会发生，但当用它们代替CTP时就不会发生。该结果表明，尽管它们在低音配对中存在歧义，但它们的行为仅作为UTP类似物。相比之下，N^4-MeCTP仅表现为CTP模拟。利用商业样品研究了典型的核苷酸氧化产物isoogtp的掺入特性。它显示没有被RNA聚合酶掺入。逆转录模板中类似物的性质。以上发现使我们能够制造出U或C完全被类似物取代的RNA。因此，我们制备了具有类似物的RNA，并将其作为AMV逆转录酶逆转录的模板。将得到的cDNA扩增并用DNA测序仪进行分析。分析表明，N^4-羟基胞苷的C和T几乎相等。这表明这种类似物可能在逆转录过程中引起突变。类似物的抗hiv病毒活性。一些类似物被发现是非常有希望的。进一步的研究仍在继续。对RNA病毒及其聚合酶的影响。研究了三磷酸类似物对脊髓灰质炎RNA聚合酶的作用。壳聚糖-三磷酸配合物的使用。我们发现用这种方法制备的壳聚糖-三磷酸配合物不够稳定。目前正在对协议进行改进。