title: Molecular beacon based bioassay for highly sensitive and selective detection of nicotinamide adenine dinucleotide and the activity of alanine aminotransferase.
abstract: We have developed a new approach to detect nicotinamide adenine dinucleotide (NAD(+)) with high specificity and sensitivity using molecular beacons (MBs) and employed it in the investigation of NAD(+) related biological processes, such as calorie restriction and alanine aminotransferase (ALT) activation. The E. coli DNA ligase would catalyze the ligation of two short oligonucleotides that complement with an MB only in the presence of NAD(+), resulting in the opening of the MB and the restoration of fluorescent signal. Thanks to the high sensitivity of the MB probe and the fidelity of E. coli DNA ligase toward its substrates, this approach can detect 0.3 nM NAD(+) with high selectivity against other NAD(+) analogs. This novel assay can also provide a convenient and robust way to analyze NAD(+) in biological samples such as cell lysate. As NAD(+) plays an essential role in many biochemical processes, this method can be used to investigate NAD(+) related life processes. For instance, the effect of calorie restriction on the intracellular NAD(+) level in MCF7 cells has been studied using this new assay. Moreover, this approach was also successfully used to analyze the activity of ALT. Therefore, this novel NAD(+) assay holds wide applicability as an analytical tool in biochemical and biomedical research.
authors.affiliation: State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Key Laboratory for Bio-Nanotechnology and Molecule Engineering of Hunan Province, Changsha 410082, China.
authors.forename: Zhiwen
authors.initials: Z
authors.lastname: Tang
authors.forename: Pei
authors.initials: P
authors.lastname: Liu
authors.forename: Changbei
authors.initials: C
authors.lastname: Ma
authors.forename: Xiaohai
authors.initials: X
authors.lastname: Yang
authors.forename: Kemin
authors.initials: K
authors.lastname: Wang
authors.forename: Weihong
authors.initials: W
authors.lastname: Tan
authors.forename: Xiaoyuan
authors.initials: X
authors.lastname: Lv
mesh_terms: D000410:Alanine Transaminase / Q000378:metabolism*; D001483:Base Sequence; D055162:Biocatalysis; D001681:Biological Assay / Q000379:methods*; D031204:Caloric Restriction; D002457:Cell Extracts; D045744:Cell Line, Tumor; D011088:DNA Ligases / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology; D005240:Feasibility Studies; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D055029:Inverted Repeat Sequences; D009243:NAD / Q000378:metabolism*; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013997:Time Factors
chemical_list: D002457:Cell Extracts; D015345:Oligonucleotide Probes; D009243:NAD; D000410:Alanine Transaminase; D011088:DNA Ligases