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，我们已经建立了n^4-羟基辛替丁三磷酸（N^4-OHCTP），N^4-甲氧基胞替胺（N^4-moctp）和N^4^4^4-甲基辛丁二胺（N^4-MCCTP）的方法。使用这些纯样品，研究了它们掺入由T3 RNA聚合酶催化的RNA。如果存在n^4-OHCTP或N^4-MOCTP代替UTP，则进行RNA合成，但不能将其添加代替CTP。该结果表明，尽管他们在低音配对中含糊不清，但它们仅作为UTP类似物的行为。相反，N^4-Mectp仅作为CTP类似物的表现。使用商业样品研究了ISOGTP的掺入性能，这是核苷酸的典型氧化产物之一。它显示RNA聚合酶没有掺入。2。反向图中的模板中类似物的概要。above发现使我们能够使RNA完全替换为u或c，其una被类似物完全替换。因此，我们用类似物制备了RNA，并将它们用作用AMV逆转录酶进行逆转录的模板。放大产生的cDNA并用DNA测序仪分析。分析表明，n^4-羟基辛丁定为C和T几乎均等。这表明该模拟可能在转录过程中引起突变3。类似物的抗HIV病毒活性。一些类似物的塑料发现非常有希望。进一步的研究继续进行4。研究了对RNA病毒及其聚合酶的作用。研究类似物三磷酸对脊髓灰质炎RNA聚合酶的作用。5。我们发现，通过报告方法制造的壳聚糖 - 三磷酸酯复合物的使用不够稳定。协议的改进目前正在进行中。