利用以阳离子共轭聚合物为能量供体的荧光共振能量转移（FRET）策略和滚环扩增放大技术, 建立了一种新型的microRNA（miRNA）检测方法。 阳离子共轭聚合物采用聚［（9,9-双（6’-N,N,N-三乙基铵）己基）亚芴基亚苯基二溴化物］（PFP）。 PFP是一种由大量吸光单元共轭而成的阳离子聚合物, 具有独特的光捕获和荧光增强性能, 可以和带有负电荷的DNA通过静电作用相互结合。 SG是一种能够结合于所有双链DNA双螺旋小沟区域的染料, 其在游离状态下, 荧光微弱, 但一旦与双链DNA结合后, 荧光会大大的增强。 首先, 设计了一条可与目标分子特异性杂交的锁式探针和与RCA产物序列互补的DNA链。 当体系中存在miRNA时, 在T4 DNA连接酶作用下, 锁式探针连接成环； 随后, 在phi29 DNA聚合酶和dNTPs共同作用下, 在miRNA的3’端滚环扩增出一条与锁式探针序列互补的长单链DNA, 所得产物与互补DNA链杂交形成双链DNA（dsDNA）。 此时SG作为FRET受体掺入其中, 形成SG-dsDNA共同体。 随后, SG-dsDNA与PFP因静电相互作用而紧密接近, 由于PFP的发射光谱与SG的激发光谱有重叠, 因此二者之间可以发生FRET现象。 反之, 当体系中不存在miRNA时, 挂锁探针则无法连接成环, 阻止了扩增反应的进行及其产物与互补DNA链的杂交反应。 加入SG后, 由于SG与单链DNA的结合能力很弱, SG则游离于溶液中, 不会与PFP发生有效的FRET。 因此目标分子的浓度与体系的FRET效率直接相关。 以let 7a作为待测miRNA分子, 在0.05～5 nmol·L-1的范围内, let 7a的浓度与从反应体系测得的FRET效率（I520/I423）成正比。 同时以无PFP参加的检测方案作为对比实验, 证明了PFP确实具有提高灵敏度的作用。 另外, 以四种同族miRNA分子及两种其他miRNA分子作为干扰物质对方法的特异性进行了考察, 发现除了两种与目标分子序列高度相似的物质存在干扰外, 其他物质几乎不产生信号。 利用该方法对细胞总RNA提取液中let 7a的含量及其加标含量进行了检测, 测量所得回收率基本令人满意。 所建立的方案不需要荧光标记探针, 有效降低了检测成本, 简化了操作步骤, 在与miRNA相关的疾病诊断领域具有一定的应用前景。

A label-free analysis strategy of miRNA is designed using the rolling circle amplification (RCA) and fluorescence resonance energy transfer (FRET) with cationic conjugated polymer (CCP) as donor. In this strategy, the cationic poly ［(9, 9-bis(6’-N, N, N-triethylammonium)hexyl) fluorenylenephenylene dibromide］ (PFP) serves as the donor of FRET and SYBR Green Ⅰ（SG）serves as the acceptor. PFP is a water-soluble π-conjugated polymer with cationic charged side chain functionalities. Its structure allows for efficient intrachain and interchain energy transfer mechanisms. It can be combined with DNA by electrostatic interaction. SG is an asymmetrical cyanine dye which preferentially binds to double-stranded DNA (ds-DNA) and stains single-stranded DNA with lower performance. The fluorescence of SG is weak in the free state, but greatly enhanced once the DNA-SG-complex is formed. Let 7a is used as the target molecule. A padlock probe matched with let 7a and the DNA probes matched with the RCA product are designed. In the presence of loret 7a, the hybridization of the padlock probe and target sequences brings two ends of padlock probe close together and can be covalently ligated into a loop in the catalyzing of T4 DNA ligase. When phi29 DNA polymerase and dNTPs are added, the rolling circle amplification of the circularized padlock probe is initiated from the target molecules and then a long single strand DNA with a lot of repetitive sequences is generated. When DNA probes and SG are added, a long ds-DNA is produced and stained by SG. The DNA-SG-complex and PFP are absorbed together through electrostatic interaction and the strong FRET from PFP to SG occurs due to the overlapping between the fluorescent emitting spectrum of the PFP and the absorption spectrum of SG. In the absence of let 7a, the padlock probe is not circularized, which induces the inhibition of rolling circle amplification and hybridization process. Therefore, the FRET also cannot occur. As a result, the let 7a can be quantitatively determined by monitoring the change of FRET signal. The results show that the let 7a concentrations in the range of 0.05～5 nmol·L-1 are linearly proportional to the detection signals. The specificity of this method is studied and the most of tested interfering substances have no influence on the test result of let 7a except let 7b and let 7c. Additionally, by the detection of let 7a concentration in the extract solution of cells, it is indicated that the strategy can be applied to the practical samples analysis. Because fluorescent labeling is not required, this strategy reduces the detection cost and simplifies the operation steps. Therefore, this protocol shows certain potential in the study of miRNA-related biological processes as well as disease diagnosis.