[Selecting and affinity analysis of DNA aptamers to MPT64 protein from Mycobacterium tuberculosis].
OBJECTIVE
To obtain DNA oligonucleotide aptamers which can specifically bind to MPT64 protein from Mycobacterium tuberculosis by SELEX technology.


METHODS
An in vitro synthesized 78 per random DNA library was subjected to 12 rounds of selection by SELEX (Systematic evolution of ligands by exponential enrichment) method against MPT64 protein. Binding of the aptamers to the protein was examined by biotin-streptavidin-horseradish peroxidase system. DNAMAN package was employed to analyze the sequences and the second structures of the aptamers. Moreover, target protein was bound to one aptamer and another aptamer modified with biotin together forming a sandwich-like complex, which was captured in microwell, to be tested in negative group including BCG and reference strains from nontuberculous mycobacteria, and positive group including H37Rv, Mycobacterium bovis reference strain, and clinical strains from Mycobacterium tuberculosis.


RESULTS
After 12 rounds of selection, high-affinity aptamers to MPT64 was obtained. The OD value at 450 nm of affinity of aptamers to MPT64 protein was from 0. 492 to 1.243, in which 73.3% was over 1.0. Pocket and stem-loops was the basis of aptamers binding to MPT64 protein by the analysis of structures,with several GC pairs among bridges between pocket and stem-loops. The analysis of the sandwich-like complex system based on two aptamers and protein showed that the positive percentage was 87. 9% in the positive group while the negative percentage was 85.7% in the negative group, with positive H37Rv and Mycobacterium bovis, and negative BCG, when the cut-off value for a positive response was 0.61 OD.


CONCLUSIONS
A set of aptamers with considerable binding affinity to MPT64 protein were successfully selected from the initial random DNA library.
Qin
Lian-Hua
LH
Shanghai Tuberculosis Key Laboratory, Shanghai Pulmonary Hospital Affiliated to Tongji University, Shanghai 200433, China.
Ma
Zhan-Zhong
ZZ
Zheng
Rui-Juan
RJ
Liu
Zhong-Hua
ZH
Lu
Jun-Mei
JM
Hu
Zhong-Yi
ZY
D000942:Antigens, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D052157:Aptamers, Nucleotide / Q000096:biosynthesis*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D015723:Gene Library; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000302:isolation & purification; D009690:Nucleic Acid Conformation; D052156:SELEX Aptamer Technique / Q000379:methods*
D000942:Antigens, Bacterial; D052157:Aptamers, Nucleotide; D001426:Bacterial Proteins; C080865:MPB64 protein, Mycobacterium