基于类修饰竞争结合和纳米免疫荧光探针识别的RNA修饰直接快速检测新方法

Detection of RNA-mediated epigenetic modifications plays an important role in precision medicine initiative. At present, the rapid and direct detection of RNA modifications encountered the following dilemma: The RNA modifications cannot be detected directly by high throughput sequencing and nucleic acid hybridization, because chemical modifications on RNA often do not change the base pairing properties of the modified bases; As the restriction of the RNA secondary structures and relative low levels of modifications in real samples, the pretreatment procedures were complicated; It is difficult to obtain good accuracy as the RNA was destroyed seriously. Aiming to these problems, we coupled the antibody and silver-nanocluster quantum dots creatively, Designed a new analogous modified DNA probes and nano-immune fluorescent probes. Explore the kinetics parameters of the proximity effects to enhance analytical performance. The developed biosensor based on analogous modifications and nano-immune fluorescent probes for RNA modifications is an original method. Multicolor quantum dots were synthesised and used to modify different antibodies, result in multiple detection was realized for different RNA modifications. This project could provide technical support for clinical application of biosensor. Moreover, it would promote the development of RNA modifications in vitro diagnostic and life sciences research.

RNA表观遗传修饰检测在精准医疗中具有重要的理论研究与临床应用价值。为了实现RNA修饰的直接快速检测，还需要解决三个关键科学问题：修饰物不改变核酸碱基性质，用单纯测序和核酸杂交的原理无法直接检测；实际样本RNA修饰的相对含量低，并且受核酸二级结构限制，样本预处理较复杂；RNA在预处理环节容易降解，影响检测结果的准确性。针对上述问题，本项目将创新性耦合抗体特异性识别和纳米荧光放大技术，巧妙设计类RNA修饰的DNA探针和纳米免疫荧光探针；探索核酸邻近效应的动力学特征，提高RNA修饰检测性能；合成并运用多色量子点标记不同抗体，实现RNA修饰的多重检测。基于类修饰竞争结合和纳米免疫荧光探针识别构建的生物传感技术平台用于临床样本中RNA修饰直接快速检测是创新性思路。该项目的实施将为生物传感器的临床应用提供全新技术平台，为开创RNA修饰在生命科学研究及体外分子诊断中的运用奠定基础。

RNA表观遗传修饰检测在精准医疗中具有重要的理论研究与临床应用价值。为了实现RNA修饰的直接快速检测，还需要解决三个关键科学问题：修饰物不改变核酸碱基性质，用单纯测序和核酸杂交的原理无法直接检测；实际样本RNA修饰的相对含量低，并且受核酸二级结构限制，样本预处理较复杂；RNA在预处理环节容易降解，影响检测结果的准确性。针对上述问题，本项目将创新性耦合抗体特异性识别和电化学技术，巧妙设计类RNA修饰的DNA探针；构建了基于类修饰DNA探针和免疫竞争法的RNA N6-甲基腺苷无标记电化学检测；设计PtCo纳米电化学信号探针，完成了基于类修饰DNA-PtCo纳米探针的RNA甲基化检测电化学生物传感器研究；根据m6A修饰RNA和未修饰RNA和类核酸探针杂交存在热力学差异，构建了一种单碱基分辨RNA甲基化特异性PCR检测新方法，整合CRISPR/Cas12a技术，实现了对低甲基化样本以及实际样本RNA的高效检测。该项目的实施将为生物传感器的临床应用提供全新技术平台，为开创RNA修饰在生命科学研究及体外分子诊断中的运用奠定基础。