Tissue-SELEX技术筛选靶向动脉粥样硬化组织的核酸适配体

Tissue-SELEX for screening nucleic acid aptamers targeting atherosclerotic tissue

摘要:

目的]采用指数富集的配体系统进化技术(SELEX)筛选并鉴定特异性结合小鼠动脉粥样硬化(As)病理组织的核酸适配体,为开发As发生早期的分子靶标及诊断试剂提供研究基础。[方法]构建库容量为10¹⁵～10¹⁶的单链DNA(ssDNA)文库,与小鼠正常血管组织切片结合-洗脱(反筛),洗脱后的文库再与小鼠As血管冰冻切片结合-洗脱(正筛)。PCR扩增正筛和反筛产物,琼脂糖凝胶电泳验证扩增产物。筛选结束后对PCR扩增产物进行T-A克隆及测序,获得核酸适配体的一级结构,Mfold在线软件预测二级结构。将筛选出的核酸适配体5′-端标记FAM荧光基团,与正筛和反筛冰冻切片结合,荧光显微镜观察荧光强度,Image Pro Plus 6.0计算相对平均荧光强度,评价核酸适配体结合的特异性。 [结果]经过8轮筛选,琼脂糖凝胶电泳成像显示正筛产物泳道出现PCR扩增产物,而反筛产物泳道并无PCR扩增产物,表明成功获得特异性结合As组织的核酸适配体；经T-A克隆及测序鉴定出5条核酸适配体,预测的二级结构均具有茎-环结构；免疫荧光染色验证5条核酸适配体与As血管均有不同程度的结合,相对平均荧光强度定量结果显示核酸适配体No.11的相对平均荧光强度数值最高,可作为候选核酸适配体用于后续研究。 [结论]成功获得特异性结合As血管的核酸适配体,为下一步筛选As发生早期的分子靶点,以及开发体内早期诊断试剂提供研究基础。

Abstract:

Aim Systematic evolution of ligands by exponential enrichment (SELEX) techniquewas employed to screen and identify nucleic acid aptamers that specifically bind to mouse atherosclerotic pathological tissues, aiming to provide a research foundation for the development of molecular targets and diagnostic reagents for early atherosclerosis. Methods A single-stranded DNA (ssDNA) library with a capacity of 10¹⁵～10¹⁶ was constructed, which was then subjected to binding-elution (negative selection) with normal mouse vascular tissue slices. The eluted library was subsequently bound to atherosclerotic tissue slices for binding-elution (positive selection). PCR was used to amplify the positive and negative screening products, and agarose gel electrophoresis was used to verify the amplified products. The ssDNA library after multiple rounds of selection was sequenced using T-A cloning and sequencing to obtain the primary structure of the nucleic acid aptamers, and the secondary structure was predicted using the Mfold online software. The selected nucleic acid aptamers were labeled with a FAM fluorescent group at the 5′- end and were bound to both positive and negative selection tissue slices, with fluorescence intensity observed under a fluorescence microscope. Image Pro Plus 6.0 was used to calculate the relative average fluorescence intensity to evaluate the binding specificity of nucleic acid aptamers. Results After 8 rounds of selection, agarose gel electrophoresis imaging showed PCR amplification products in the positive selection lanes, while no PCR amplification products were observed in the negative selection lanes, indicating the successful acquisition of a nucleic acid aptamer library that specifically binds to atherosclerotic tissues. Five nucleic acid aptamers were identified by T-A cloning and sequencing, and their predicted secondary structures all had stem-loop structures. Immunofluorescence staining verified that five nucleic acid aptamers had different degrees of binding with As blood vessels, and the quantitative results of the relative average fluorescence intensity showed that nucleic acid aptamer No.11 had the highest relative average fluorescence intensity value, which can be used as a candidate nucleic acid aptamer for subsequent research. Conclusion Specific nucleic acid aptamers that bind to atherosclerotic vesselswere successfully obtained, providing a research foundation for further screening of early molecular targets of Asand developing in vivo early diagnostic reagents. [KEY WORDS]atherosclerosis; systematic evolution of ligands by exponential enrichment; nucleic acid aptamers