A Preliminary Study On Signal Amplification Strategy Based On AP Site Regulation In Detection Of MiRNA-122 And UDG

AP sites(apurinic/apyrimidinic site,AP site)are also called abasic sites.It is caused by DNA damage and has neither purine nor pyrimidine at one location.Under physiological conditions,the human body produces about 10,000 AP sites every day.In living cells,if these AP sites are not repaired,it will prevent DNA replication,which will lead to DNA strand breaks,cell mutations,and even cell death.AP site is an important feature of base excision repair pathway.DNA glycosylase will recognize the diseased base and create an AP site,then AP endonuclease will form an incision at the AP site,and finally repair it with ligase.MicroRNA(miRNA)plays a vital role in many biological processes.Recent studies have shown that miRNA expression profiling may provide information and be used as a biomarker for the diagnosis and prognosis of diseases such as cancer and diabetes.Therefore,the development of new miRNA detection technology with selectivity and sensitivity and easy operation is of great significance for early clinical diagnosis.However,due to the short sequence and low content of miRNA,the existing detection methods usually require complex primer design and chemical modification.In order to solve the above problems,this paper proposes a technique of PCR repair with AP sites that can be triggered by miRNA to synthesize repair,thus developing a novel miRNA detection strategy called template repair PCR(TR-PCR).The detection limit of miRNA-122 is 2 amol.Uracil DNA glycosylase(UDG)is a highly conservative damage repair glycosylase.The abnormal expression of DNA glycosylase has important research value in many human diseases.However,the existing detection principles need to face complex sequence structure design or balance between multiple enzyme systems.In response to the above problems,this article replaced the thymine bases(dT)at different positions of the T7 promoter sequence with uracil bases(dU).We found that this substitution has little effect on the transcription of T7 RNA polymerase.However,when UDG is present,dU is cut to produce AP sites,which will have a great influence on transcription.Some AP sites will promote transcription,and some AP sites will inhibit transcription.Taking advantage of this feature,we have designed a new UDG detection principle that does not require complex melting chain temperature design and does not require the participation of multiple enzyme systems.The sensitivity of this method is 2.5×10^-4U/mL,and it has good specificity.It can also work normally in complex actual samples such as He La cell lysate.At present,this detection principle has not been applied to UDG activity detection.To sum up,this paper uses the effect of AP sites on DNA polymerase synthesis and the effect of T7 RNA polymerase promoter recognition to construct a sensitive and specific strategy for detecting miRNA and UDG activity.These studies may become promising technologies for highly sensitive miRNA detection and UDG activity detection.