Construction Of DNA Biosensors Based On G-Quadruplex And Enzymatic Amplification Strategies For The Detection Of Uracil-DNA Glycosylase

As an important DNA repair enzyme,uracil-DNA glycosylase?UDG?plays a crucial role in maintaining the genetic integrity of organisms.It seriously affects the repair of uracil base mutations and is closely related to lymphoma,Bloom syndrome,chemotherapy resistance and other diseases.Therefore,the monitoring of UDG activity is essential for the study of base excision repair?BER?mechanism and clinical diagnosis.As a kind of safe,simple,and sensitive biosensor,DNA fluorescence sensors have been widely used in biochemical analysis.Resonance Rayleigh scattering?RRS?has been gradually used in the construction of DNA sensors due to its ultra-sensitive properties.In this paper,a DNA biosensor based on the G-quadruplex amplification strategy and a DNA biosensor based on an Exo ?-assisted multiplex amplification for the detection of active UDG were constructed.In addition,since the unique G-wires can significantly enhance the resonance Rayleigh scattering signal,we constructed an Exo ?-assisted RRS biosensor for active UDG detection for the first time based on the G-wire structure.The main contents are as follows:1.Linked bridge hybridizing-induced split G-quadruplex biosensor and its application to uracil-DNA glycosylase detectionBy linked bridge hybridizing-induced split G-quadruplex?SQ?,we demonstrate here the construction of a simple and sensitive DNA biosensor for the detection of UDG activity.The satisfactory split G-quadruplex sequences?SQS?were successfully selected to be the ingredients of SQ formation.In this work,UDG recognized and removed the uracil bases from the stem of hairpin DNA?HP?.And then,HP with a low melting temperature hybridized with designed SQS,forming a three-way DNA structure with a SQ.With the addition of Thioflavin T,a dramatical enhancement of fluorescence intensity was presented due to the G-quadruplex/Thioflavin T complex formation.The detection limit was as low as 7.8×10^-3 U/mL.And we also successfully investigated the performance of UDG activity in the HeLa cell lysate.This optical DNA biosensor with the merits of being simple,rapid,and economical was flexibly suitable for not only active UDG assay but also diverse target detection by adjusting the recognition region of the HP.2.Sensitive label-free resonance Rayleigh scattering method-based dual amplification strategy for the active uracil-DNA glycosylase assayHere,we proposed a RRS strategy for sensitively detecting the active UDG based on a dual amplification DNA biosensor without label under the catalysis of exonuclease ??Exo ??.In our study,a double-stranded DNA complex S1-S2 containing triggers and peculiar uracil bases was employed.To achieve the dual amplification strategy,we adopted hairpin probes HP1 and HP2.The unique hairpin probes can partially hybridize with S1 and S2,respectively.With the excision reaction of UDG,the uracil bases were excised from S2 in double-stranded S1-S2,resulting in a lower melting temperature of the S1-S2.Then,the S1-S2 was dissociated to single-stranded S1 and S2 with abasic sites.Subsequently,the liberated strands separately hybridized with hairpins to initiate the Exo ?-catalyzed dual amplification.In the presence of K⁺,countless c-myc sequences which were produced by the amplification reaction formed G-quadruplex structures.Finally,with the addition of Mg²⁺,long and continuous G-wire structures were obtained,causing an obvious enhancement of RRS intensity.The proposed RRS DNA biosensor obtained the limit of detection as low as 1.0×10^-5 U/mL for the active UDG assay.Moreover,qualitative analysis of the active UDG was successfully achieved in HeLa cells lysate.The DNA biosensor is a potential tool to be used in UDG clinical diagnosis or functional study.3.Sensitive detection of active uracil-DNA glycosylase via an exonuclease ?-assisted cascade multi-amplification fluorescence biosensorHere we demonstrated a sensitive cascade multi-amplification fluorescence strategy for the active UDG assay with the help of exonuclease ??Exo ??.Under the cleavage reaction of UDG,the double-stranded DNA?dsDNA?containing peculiar uracil bases separated into two dissociative single-stranded DNA,which would separately hybridize with two cyclic hairpin probes to launch the Exo ?-assisted dual cycles.The hairpin DNA modified with fluorophore and quenching group was employed as the signal probe.After the dual cycles,countless same short DNA fragments were generated to hybridize with signal probes,initiating a new Exo ?-assisted cyclic amplification and releasing numerous single-stranded DNA which only carried fluorophore.Thus,the fluorescence intensity of the detection system was enhanced.This study obtained the detection limit low to 2.4×10^-4 U/mL for detecting the UDG activity.And it performed well practical applicability by analysis the HeLa cell lysate and satisfactory selectivity,pro viding a potential method for clinic diagnosis and functional study of UDG activity.