Detection Of Important Biomolecules Using Molecular Beacon

The quantitative analysis of the biomolecules associated with the key function of living organisms are utmost important in early diagnosis and therapy of genetic diseases. In this work, by using specifity, real-time and simplicity of molecular beacons and coenzyme-dependence of DNA ligase, novel detection methods for adenosine triphosphate (ATP), nicotinamide adenine dinucleotide phosphate (NADP), creatine kinase (CK) and protein kinase A (PKA) were developed:1. Detection of creatine kinase activity using molecular beaconA novel method for the determination of CK activity based on ATP-dependent T4 DNA ligase has been developed. The ATP was produced from adenosine 5'-diphosphate (ADP) and creatine phosphate (CrP) catalyzed by CK. Then T4 DNA ligase catalyzed the ligation reaction with ATP resulting the formation of whole-match DNA, which caused conformation changes of molecular beacon. The increase of fluorescent intensity of molecular beacon was related to the concentration of CK. This assay could determine CK in the range of 1 ~ 50 U/L with a detection limit of 0.64 U/L. Moreover, the method was shown to be suitable for the sensitive detection of CK inhibitors.2. Detection of protein kinase A using molecular beaconPKA was detected by quantifying the ATP after the protein kinase reaction. ATP assay was achieved by using T4 DNA ligase and molecular beacon. This assay could determine PKA in the range of 12.5 ~ 150 nM with a detection limit of 1.25 nM. Moreover, the assay was available to investigate the effect of genistein on PKA. It was a universal, non-radioisotope and homogeneous method for PKA assay.3. An enzymatic cycling and signal amplification assay for ATP using molecular beaconA sensitive assay for ATP was developed by enzymatic cycling and signal amplification assay. T4 DNA ligase consumed ATP and produced AMP, which was phosphorylated to ATP with 2'-deoxycytidine 5'-triphosphate (dCTP) as the phosphate donor catalyzed by adenylate kinase and nucleoside-diphosphate kinase. The ATP was detected by molecular beacon and T4 DNA ligase mentioned above. With the cycling of ATP→AMP→ATP, more and more molecular beacons were opened. This assay could determine ATP in the range of 0.01 ~ 10 nM with a detection limit of 5 pM. The sensitivity of the method developed here is comparable with bioluminescence.4. A high-sensitive method for the detection of NADP using molecular beaconNADP assay was completed based on high-dependence of E. coli DNA ligase on coenzyme, nicotinamide adenine dinucleotide (NAD). First, NADP was transformed to NAD by alkaline phosphatase (CIAP), and then NAD product was introduced into molecular beacon system and quantified. NADP concentration was represented by the initial enhancement rate of fluorescence intensity. This assay could determine NADP in the range of 5 ~ 200 nM with a detection limit of 2 nM. Compared with current assay methods, this approach was convenient, quick and highly sensitive.