Self-replicating, self-amplifying probes for detection of disease-related nucleic acid sequences

核酸序列特异性检测方法在现代分子生物学中具有重要意义。酶结扎通常用于诊断目的，尽管它们显示出一些局限性。根据所使用的酶，RNA和修饰的核酸的连接通常比未修饰的DNA效率低。此外，酶连接反应很难在体内进行。虽然非酶探针是可用的，但它们缺乏信号放大。最近，Kool及其同事开发了由修饰DNA组成的探针，通过对单次错配具有高特异性的自连接机制，用于有效检测DNA和RNA靶序列。这个非酶促反应显示出一些信号放大。利用这些自结扎探针，已经有可能通过靶向核糖体RNA原位检测大肠杆菌。该研究项目旨在开发一种新的自连接探针，用于检测固定细胞和活细胞中含量较低的RNA甚至DNA。一个新的探针设计应该防止产品抑制的常见问题，使提高周转。此外，通过使自我连接呈指数级发生，应该获得更高的灵敏度，从而能够检测致病菌的痕迹，也可以感知和定位人类细胞中与疾病相关的DNA和RNA序列，包括单核苷酸多态性、致癌基因和错误剪接事件。

Methods for the specific detection of nucleic acid sequences are of high importance in modern molecular biology. Enzymatic ligations are often used for diagnostic purposes, although they display some limitations. Depending on the enzymes used, ligation of RNA and modified nucleic acids is often inefficient compared to unmodified DNA. In addition, enzymatic ligation reactions are difficult to carry out in vivo. Although nonenzymatic probes are available, they lack signal amplification. Recently, Kool and co-workers have developed probes consisting of modified DNA, which were used to efficiently detect DNA and RNA target sequences by a self-ligation mechanism with high specificity against single mismatches. This nonenzymatic reaction shows some signal amplification. By utilizing these self-ligation probes, it has been possible to detect Escherichia coli by targeting ribosomal RNA in situ. The proposed research project aims at developing a new class of self-ligating probes for detection of less abundant RNA and even DNA in fixed and living cells. A novel probe design should prevent the common problem of product inhibition, enabling enhanced turnover. Furthermore, by rendering the self-ligation to occur exponentially, increased sensitivity should be gained, enabling detection of traces of pathogenic bacteria as also to sense and localize disease-related DNA and RNA sequences in human cells including single nucleotide polymorphisms, oncogenes and mis-splicing events.