靶向细菌16S rRNA核酸抗菌素的初步研究
发布时间：2019-05-06 09:24
【摘要】：目的:反义技术的发展为研发新一代抗菌药物提供了新思路。本研究以大肠杆菌16S rRNA为药物作用靶标,运用本实验室建立的MAST技术平台筛选其反义核酸作用靶点;通过体外和胞内实验对筛选靶点的有效性进行验证,以期获得能高效结合反义寡核苷酸的靶点序列;根据有效作用靶点合成硫代反义寡核苷酸研究其抑菌效果,为研发新一代核酸抗菌药物进行有益探索。方法:通过在转录16S rRNA过程中引入生物素标记的UTP,将其固定到亲和素磁珠上,保持RNA的自然折叠状态。然后与寡核苷酸文库杂交,筛选能结合的文库序列从而阐明靶点,该方法即为MAST技术(mRNA accessible site tagging)。运用该技术筛选获得16S rRNA6个反义寡核苷酸结合靶点。根据靶点,设计并合成6条靶点特异的反义寡核苷酸和5条10-23型脱氧核酶(DNAenzyme)。采用反义寡核苷酸依赖RNase H切割技术对6条反义寡核苷酸体外对16S rRNA的结合能力进行检测,并采用10-23型脱氧核酶催化切割技术对其中5条脱氧核酶的体外结合切割活性进行检测。经体外验证,所筛6个靶点中5个靶点为16S rRNA可及位点,其中靶点Ⅴ(907-926nt)结合活性最好。然后,构建原核表达载体用IPTG诱导表达单倍体锤头状核酶(sRZ)和双倍体锤头状核酶(dRz),利用表达的核酶胞内特异降解16S rRNA抑制大肠杆菌生长,对靶点Ⅴ胞内有效性进行验证。针对体外和胞内验证有高效结合活性的靶点Ⅴ,合成硫代反义寡核苷酸与通透性好的大肠杆菌SM101温育,检测其对大肠杆菌生长的抑制效果,进行核酸抗菌素的初步研究。结果:运用MAST技术筛选获得位于16S rRNA 179-198、446-465、497-516、887-906、907-926、1236-1255nt6个靶点(Ⅰ-Ⅵ);反义寡核苷酸依赖RNase H切割活性分析结果显示,靶点Ⅱ-Ⅵ为反义寡核苷酸体外高效结合靶点;脱氧核酶催化切割活性分析结果显示,靶点Ⅴ体外活性最为显著;胞内验证结果表明,针对靶点Ⅴ的单倍体和双倍体核酶对大肠杆菌生长均有抑制效果,且双倍体核酶效果更为显著;针对靶点Ⅴ的硫代反
[Abstract]:Objective: the development of antisense technology provides a new idea for the development of a new generation of antibacterial drugs. In this study, Escherichia coli 16s rRNA was used as the target of drug action, and the target of antisense nucleic acid was screened by using the MAST technology platform established in our laboratory. The effectiveness of screening target was verified by in vitro and intracellular experiments in order to obtain the target sequence which could bind antisense oligodeoxynucleotides efficiently. The antibacterial effect of antisense thioate oligodeoxynucleotides (ASODN) was studied according to the effective target, and a useful exploration for the development of new generation of nucleic acid antibacterial drugs was carried out. Methods: biotin-labeled UTP, was introduced into the 16s rRNA transcription process to immobilize it to avidin magnetic beads to maintain the natural folding state of RNA. And then hybridized with the oligonucleotide library to screen the binding library sequence to clarify the target. This method is called MAST technique (mRNA accessible site tagging). 16s rRNA6 antisense oligodeoxynucleotides (ASODN) binding targets were screened by this technique. According to the target, six target specific antisense oligonucleotides and five 10-23 deoxyribozyme (DNAenzyme). Were designed and synthesized. The binding ability of six antisense oligodeoxynucleotides to 16s rRNA in vitro was detected by RNase H cleavage technique. The in vitro binding activity of 5 deoxyribozymes was detected by 10-23 type deoxyribozyme catalytic cleavage technique. Five of the six screened targets were 16s rRNA accessible sites, among which the binding activity of target 鈪，
本文编号：2470069
[Abstract]:Objective: the development of antisense technology provides a new idea for the development of a new generation of antibacterial drugs. In this study, Escherichia coli 16s rRNA was used as the target of drug action, and the target of antisense nucleic acid was screened by using the MAST technology platform established in our laboratory. The effectiveness of screening target was verified by in vitro and intracellular experiments in order to obtain the target sequence which could bind antisense oligodeoxynucleotides efficiently. The antibacterial effect of antisense thioate oligodeoxynucleotides (ASODN) was studied according to the effective target, and a useful exploration for the development of new generation of nucleic acid antibacterial drugs was carried out. Methods: biotin-labeled UTP, was introduced into the 16s rRNA transcription process to immobilize it to avidin magnetic beads to maintain the natural folding state of RNA. And then hybridized with the oligonucleotide library to screen the binding library sequence to clarify the target. This method is called MAST technique (mRNA accessible site tagging). 16s rRNA6 antisense oligodeoxynucleotides (ASODN) binding targets were screened by this technique. According to the target, six target specific antisense oligonucleotides and five 10-23 deoxyribozyme (DNAenzyme). Were designed and synthesized. The binding ability of six antisense oligodeoxynucleotides to 16s rRNA in vitro was detected by RNase H cleavage technique. The in vitro binding activity of 5 deoxyribozymes was detected by 10-23 type deoxyribozyme catalytic cleavage technique. Five of the six screened targets were 16s rRNA accessible sites, among which the binding activity of target 鈪，
本文编号：2470069
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