适配体分子探针的信号放大新策略研究

Aptamers are single-stranded DNA or RNA molecules and selected from random oligonucleotide pools by a process called Systematic Evolution of Ligands by Exponential Enrichment (SELEX).The specificity and high affinity of aptamers to a wide variety of targets, coupled with the simple synthesis, thermal stability, ease of design and modification, make aptamers highly suitable for development as biomolecular probes.It is an urgent and significant task to develop highly sensitive detection methods for targets in the fields of analytical chemistry and biomedicine. For example, the early diagnosis of cancers is highly dependent on the development of a highly sensitive method for cancer biomarkers.Here, we will study the new stratgies for signal amplification of aptamer molecular probes. The specific binding properties of aptamers with their targets will be transducted to the amplified biosensing events of deoxyribozymes. Three strategies for probe signal amplification will be investigated. 1) rational design of aptazymes based on cocaine, ATP atamers and 8-17 deoxyribozyme.An aptazyme is a known aptamer sequence connected to a known deoxyribozyme sequence. In the absense of target analyte for the aptamer, the enzymatic activity can not be expressed due to inappropriate structrual folding. The presence of the target causes adaptive folding of the aptamer, which leads to the structural formation of the catalytic domain and its activation. We will also run the direct SELEX to get cocaine or ATP responsed aptazymes. 2)cascading the binding of aptamer-target with the enzymatic reaction of 8-17 deoxyribozyme. This design uses a short DNA to form duplex with aptamer, and target recognition will induce the structure-switching dissociation of short DNA. By linking this short DNA to the acitivation of downstream 8-17 deoxyribozyme, the binding of target with its aptamer is converted to the enzymatic amplification reaction of deoxyribozyme. 3) cascading upstream aptazyme with downstream 8-17 deoxyribozyme. A communication single strand DNA will be design, which acts as both the cleavable substrate of upstream aptazyme and the inhibitor of downstream deoxyribozyme. By this way, the activation of upstream aptazyme through the binding of target will cleave the commnication DNA molecule, then lead the activation of downstream deoxyribozyme. This system takes advantage of two enzymatic amplification reactions. We also investigate the ways to improve the selectivity of our developed strategies of probe signal amplification. The general mechnism of successful design of each of these probe amplification strategies will also be studied.

面向分析化学和生物医学等领域对进一步提高靶分子检测灵敏度的重大需求，本项目研究适配体分子探针的信号放大新策略。重点研究如何将靶分子和适配体的识别转化成剪切型8-17脱氧核酶的酶放大反应信号。具体研究如下三种策略：1）用理性设计的方法构建适配体与8-17脱氧核酶的核酸嵌合体，通过适配体与靶分子的识别作用，导致嵌合体构象变化从而诱导脱氧核酶的激活；同时进行适配体调控的剪切型脱氧核酶的直接筛选，以期获得相应的对靶分子响应的嵌合体。2）级联适配体和靶分子的识别反应与8-17脱氧核酶的反应，通过识别诱导解离释放短链DNA片段，此片段进而激活下游的处于失活状态的8-17脱氧核酶。3）建立上游和下游剪切型脱氧核酶的双酶反应的级联系统，通过双酶的双重放大作用，来显著提高对靶分子的检测灵敏度。并对如何提高信号放大系统对靶分子识别的选择性进行研究。进而探讨这三种分子探针信号放大策略的构建规律和内在机理。

适配体是利用指数富集的系统进化技术(SELEX)从随机核酸文库中体外筛选得到的单链DNA或者RNA分子。适配体可以特异性识别各种各样的靶标物质，并且具有易合成和修饰，热稳定性等优点，非常适合成为分子探针。为构建高灵敏，同时具有经济高效、操作简单等优势的基于适配体的检测方法，以满足分析化学和生物医学等领域对靶分子检测灵敏度、经济高效的需求，本项目利用DNA分子构象易于调控的特点，研究了适配体分子探针的信号放大策略。发展了多种信号放大策略：1）用理性设计的方法构建适配体与8-17脱氧核酶的核酸嵌合体，通过适配体与靶分子的识别作用，导致嵌合体构象变化从而诱导脱氧核酶的激活。2）级联适配体和靶分子的识别反应与8-17脱氧核酶的反应，通过识别诱导解离释放短链DNA片段，此片段进而激活下游的处于失活状态的8-17脱氧核酶。3）建立上游和下游剪切型脱氧核酶的双酶反应的级联系统，通过双酶的双重放大作用，来显著提高对靶分子的检测灵敏度。4）制备DNA水凝胶并包过氧化物酶，在DNA结构中引入适配体，通过靶分子识别控制释放酶分子，从而催化下游反应，产生信号变化，进而实现靶分子信号放大的超灵敏检测。并对如何提高信号放大系统对靶分子识别的选择性进行研究。进而探讨这几种分子探针信号放大策略的构建规律和内在机理。