没有限制的限制性内切酶-镧系金属配合物为辅基的脱氧核酶的筛选

Restriction enzymes play a crucial role in the model molecular biology and biotechnology. They can recognize and cleave specific DNA base sequences, allowing gene addition, mutation and deletion to be carried out. Current restriction enzymes can only recognize four to eight base pairs. However, to treat human disease and to answer many fundamental biological questions, tools are needed to cleave at arbitrary positions in the human chromosome. This requires restriction enzymes that can recognition at least 16 base pairs. DNAzymes and aptamers are new functional nucleic acids that were discovered in the past twenty years. Herein we propose to use in vitro selection as the major tool to isolate DNAzymes that utilize lanthanide metal complexes as cofactors for the hydrolysis of DNA. The DNA scaffold can specifically recognize any DNA sequence of choice, and lanthanides are known to have DNA hydrolysis activity. Thus, it might be possible to obtain restriction enzymes without restriction. We will fully characterize these new DNAzymes in terms of biochemical, biophysical and bioinorganic properties and study the role of metal ions. We will also perform initial studies on using these enzymes for manipulation of plasmid DNA and for applications in nanotechnology.

限制性内切酶是现代分子生物学中非常重要的一类酶。它们能切割特定的DNA序列，进而能实现基因的加入，突变和删除等操作。目前酶的特异性仅局限于4-8个碱基对，而对很多人类疾病的治疗和基本生物问题的探索则需要对人类染色体DNA进行任意切割，这需要能识别16个碱基对以上的酶。核酸适配体和核酶是近20 年内发现的功能化的核酸。在此我们提出一个基于核酸的新思路，即用DNA作为酶的骨架，用镧系金属离子配合物做辅基，用体外筛选作为主要技术手段，获得高活性和具有普适性的酶。用DNA 做骨架可以识别任意的DNA序列，就解决了蛋白质普适性差的问题。镧系金属离子能够非特异性切割DNA。在酶骨架的引导下，则有可能实现特异性切割。这样就有可能解决普适性和特异性之间的矛盾。我们将对筛选出的酶进行全面的生物化学表征，分析其生物无机化学上的意义和机理，探求金属离子的作用，并初步探索其对质粒DNA的操作和在纳米技术中的应用。

脱氧核酶(DNAzyme，DNA酶)是以金属离子为辅酶的具催化功能的DNA单链，其活性具有高度金属离子依赖性和选择性，可用于金属离子的特异性检测。本研究筛选及表征了多个具有高金属离子特异性及高活性的DNA酶（如Ce13d、EtNa、NaA43、Ce5、17EV1等），在生物物理/化学表征的基础上，明确DNA酶与金属离子识别的分子机制及金属离子在催化反应中的作用，构建了DNA酶生物信标用于金属离子检测，包括Cr3+、Hg2+、Ag+、Na+、Mg2+及Ca2+等。实验结果为DNA酶在生物无机化学，环境重金属污染检测、医学生理指标筛查等领域的应用提供实验依据。同时，研究了DNA酶在体内应用的可行性及与纳米材料的键合，为DNA酶在疾病的诊断与治疗等体内应用提供基础。