单标记荧光共振能量转移法用于解螺旋酶的检测

Single marker fluorescence resonance energy transfer method for the detection of helicase

研究单标记荧光共振能量转移法用于检测丙肝解螺旋酶.设计一段DNA双螺旋片段,其中一条DNA链的3’末端标记荧光基团,另一条链的5’末端以多个G碱基作为猝灭基团.当解旋酶不存在时,DNA双螺旋结构稳定,由于荧光共振能量转移作用,此时体系荧光较弱.加入解旋酶后,由于酶对双螺旋DNA有特异性解旋作用,使得双链DNA解离,导致末端标记的荧光基团与另一条链的G碱基远离,从而使体系的荧光增强.最佳实验条件下,解螺旋酶的浓度在1 nmol·L^(-1)~0.5μmol·L^(-1)范围内与503 nmol·L^(-1)处的荧光强度呈良好的线性关系,检测限达到0.3 nM,该体系可以用于丙肝解旋酶的测定.

To study the single marker fluorescence resonance energy transfer was method used in the detection of Helicase of Hepatitis C virus. In these assays, a DNA double helix fragment was designed, in which the 3＇ end of one DNA strand was labeled with fluorophore and the 5＇ end of the other strand was designed with a multiple of G bases as a quenching group. In the absence of helicase, fluorescence of fluorophore is quenched by nearby guanine bases. In contrast, the binding of helicase and duplex will destruct the duplex and dissociate the duplex, which separates fluorophore from guanine bases and increases the fluorescence signal. Thus, a new continuous fluorescence assay method was developed based on fluorescence resonance energy transfer for the monitoring of helicase activity. Fluorescence intensity increased with helicase concentration. In the best condition, there was a good linear relationship between the concentrationof helicase in the range of 1 nmol · L-1-0.5 μmol · L-1 and the fluorescence intensity at 503 nmol · L-1. The detection limit for helicase was calculated to be 0.3 nmol · L-1 , this system can be used in tile determination of helicase.