Influence of ionic strength, pH and aptamer configuration for binding affinity to thrombin

被引:253
作者
Hianik, Tibor [1 ]
Ostatna, Veronika [1 ]
Sonlajtnerova, Michaela [1 ]
Grman, Igor [1 ]
机构
[1] Comenius Univ, Fac Math Phys & Comp Sci, Biophys Lab, Bratislava 84248, Slovakia
关键词
DNA aptamers; thrombin; electrochemical indicators; methylene blue; QCM; dendrimers;
D O I
10.1016/j.bioelechem.2006.03.012
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We used the methods of electrochemical indicators and the quartz crystal microbalance (QCM) for detection of thrombin-aptamer interactions. We analyzed how the method of immobilization of aptamer to a solid support, the aptamer configuration as well as variation in ionic strength and pH will affect the binding of thrombin to the aptamer. The immobilization of aptamer by means of avidin-biotin technology revealed best results in sensitivity in comparison with immobilization utilizing dendrimers of first generation and in comparison with chemisorption of aptamer to a gold surface. Linear and molecular beacon aptamers of similar structure of binding site revealed similar binding properties to thrombin. Increased concentration of NaCl resulted in weakening of the binding of thrombin to the aptamers, probably due to shielding effect of Na+ ions. The binding of the thrombin to the aptamer depends on electrolyte pH, which is presumably connected with maintaining the three dimensional aptamer configuration, optimal for binding the protein. (c) 2006 Elsevier B.V All rights reserved.
引用
收藏
页码:127 / 133
页数:7
相关论文
共 23 条
[1]  
BERG W, 1979, THROMB HAEMOSTASIS, V42, P972
[2]  
BRETT CMA, 1993, ELECT PRINCIPLES MET
[3]   Detecting immobilized protein kinase C isozymes with RNA aptamers [J].
Conrad, R ;
Ellington, AD .
ANALYTICAL BIOCHEMISTRY, 1996, 242 (02) :261-265
[4]   Synthesis and radioiodination of a stannyl oligodeoxyribonucleotide [J].
Dougan, H ;
Hobbs, JB ;
Weitz, JI ;
Lyster, DM .
NUCLEIC ACIDS RESEARCH, 1997, 25 (14) :2897-2901
[5]   INVITRO SELECTION OF RNA MOLECULES THAT BIND SPECIFIC LIGANDS [J].
ELLINGTON, AD ;
SZOSTAK, JW .
NATURE, 1990, 346 (6287) :818-822
[6]   Detection of aptamer-protein interactions using QCM and electrochemical indicator methods [J].
Hianik, T ;
Ostatná, V ;
Zajacová, Z ;
Stoikova, E ;
Evtugyn, G .
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 2005, 15 (02) :291-295
[7]   Folding of the thrombin aptamer into a G-quadruplex with Sr2+:: Stability, heat, and hydration [J].
Kankia, BI ;
Marky, LA .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 2001, 123 (44) :10799-10804
[8]   An aptamer-based quartz crystal protein biosensor [J].
Liss, M ;
Petersen, B ;
Wolf, H ;
Prohaska, E .
ANALYTICAL CHEMISTRY, 2002, 74 (17) :4488-4495
[9]   3-DIMENSIONAL STRUCTURES OF AVIDIN AND THE AVIDIN-BIOTIN COMPLEX [J].
LIVNAH, O ;
BAYER, EA ;
WILCHEK, M ;
SUSSMAN, JL .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1993, 90 (11) :5076-5080
[10]   Aptamer-based biosensor arrays for detection and quantification of biological macromolecules [J].
McCauley, TG ;
Hamaguchi, N ;
Stanton, M .
ANALYTICAL BIOCHEMISTRY, 2003, 319 (02) :244-250