Applications Of Allosteric Aptamer Probe To Sensitive Detection Of DNA Kinase And Phosphatase

Aptamer is single-stranded oligonucleotide which can bind to target molecule with high specificity and affinity and obtained by Systematic Evolution of Ligands by Exponential Enrichment(SELEX).Taking advantage of the high specificity and affinity of aptamer and biomimetic allosteric effect in nature,allosteric aptamer probe(AAP)is a type of DNA molecular probe designed for high sensitive detection of a wide variety of targets including ions,small organic molecules,proteins,and even cells and tissues.Allosteric aptamer probe possesses the merits of high sensitivity,high specificity,high reaction rate and hardly influenced by complex system.And it can be used to detect a variety of targets with different customed designs.With high compatibility,allosteric aptamer probe has a wide range of applications.Herein,we developed the traditional hairpin allosteric molecular beacon,and designed two novel allosteric aptamer probes for high sensitive detection of T4 Polynucleotide Kinase(T4 PNK)and Alkaline Phosphatase(ALP),which proved that novel allosteric aptamer probe had great prospect of applications and values of clinical diagnosis.The work of this thesis mainly includes two aspects.Firstly,we desined a novel allosteric aptamer probe which consisted of two parts for the detection of T4 PNK.The first part is a fluorophore-labeled streptavidin(SA)aptamer,and the other is its full length of complementary DNA(cDNA).When T4 PNK is of precense in the system,the cDNA will be phosphorylated,inducing the degradation of cDNA by lambda exonuclease(X exo).Subsequently,the secondary structure of SA aptamer is rebuilt so that the fluorophore-labeled aptamer can bind to SA beads.As a consequence,T4 PNK can be quantitatively detected with high sensitivity by monitoring the fluorescence intensity of SA beads.The lowest detection concentration of T4 PNK can reach 0.01 U/mL.Then we focused on ALP which has totally the opposite function of T4 PNK in biological systems.We adopted an analogous scheme for the design of AAP and detection of ALP.The AAP of ALP is composed of an SA aptamer and its 5'-phosphorylated cDNA.When ALP is of absence in the system,the cDNA w ill be degradated by ? exo,which will induce the allosteric effect of the AAP and the fluorescence intensity of aptamer-bound SA beads will enhance.While if ALP exists,the 5'-P04 of cDNA will be dephosphorylated,so that the allosteric effect will be inhhibited.Thus the concentration of ALP can be determined by the decreasing of fluorescence intensity of the SA beads.The lowest detection concentration of ALP can reach 0.012 U/mL.