5'核酸外切酶特异性荧光探针的设计和应用

根据5'核酸外切酶与底物间相互作用的特点,设计开发了对其具有高选择性、高灵敏度的DNA荧光探针。该探针对5'外切酶的线性响应范围为0.005~0.1 U/m L,检测限为0.005 U/m L。方法可用于人血清样品中5'外切酶的一步快速测定,测得正常人血清中5'外切酶的含量为0.09±0.01 U/m L,回收率为100%±5%(n=3)。该研究为实时监测生物样品中5'核酸外切酶的含量变化提供了有力的工具,也将有助于深入研究核酸损伤的修复途径。

A Highly Specific and Sensitive Fluorescent Probe for the Measurement of 5'Exonucleases in Human Serum Samples

A unique DNA fluorescent probe has been developed for the measurement of 5'exonuclease with high selectivity and sensitivity.The probe(P-6) had a hairpin structure with a 5-nt overhang at the 3'end and a phosphate group at the 5'end.The fluorophore and quencher were labeled at the first and 10th nucleotide from the 5'end.Besides,an internal loop formed by three consecutive mismatched base pairs was introduced in the stem structure of the probe to prevent the degradation by DNase I.It has been experimentally proved that the probe had an excellent selectivity toward 5'exonucleases over other nucleases.Under the optimized reaction conditions,the probe showed a linear working range of 0.005-0.1 U/mL with a detection limit as low as 0.005 U/mL.The method allowed for rapid detection of the activity of 5'exonucleases in serum samples within one step without the need of sample pretreatment.The amount of 5'exonuclease in normal human serum was found to be 0.09± 0.01 U/mL and the recovery was 100％±5％ (n=3).The method provides a powerful tool for real-time monitoring of the variation of 5'exonuclease in biological samples and will also be helpful for further investigation of the DNA damage repair mechanisms in different organisms.