Photoaptamer heterodimeric constructs as a new approach to enhance the efficiency of formation of photocrosslinking with a target protein

Rakhmetova S.Yu.1, Radko S.P.1 , Gnedenko O.V.1, Bodoev N.V.1, Ivanov A.S.1, Archakov A.I.1

1. V.N. Orekhovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences
Section: Experimental/Clinical Study
DOI: 10.18097/pbmc20105601072      UDK: 577.113.6      PubMed Id: 21328912
Year: 2010  Volume: 56  Issue: 1  Pages: 72-81
Using two DNA aptamers selectively recognizing anion-binding exosites 1 and 2 of thrombin as a model, it has been demonstrated that their conjugation by a poly-(dT)-linker (ranging from 5 to 65 nt in length) to produce aptamer heterodimeric constructs results into affinity enhancement. The apparent dissociation constant (Kdapp) measured at the optical biosensor Biacore-3000 for complexes of thrombin with the heterodimeric constructs reached minimum values (Kdapp = 0,2-0,4 nМ) which were approximately 30-fold less than for the complexes with the primary aptamers. A photoaptamer heterodimeric construct was designed connecting photoaptamer and aptamer sequences with the poly-(dT)-linker of 35 nt long. The photoaptamer used could form photo-induced cross-links with the exosite 2 of thrombin and the aptamer used could bind to the exosite 1. The measured value of Kdapp for the photoaptamer construct was approximately 40-fold less than that for the primary photoaptamer (5,3 and 190 nM, respectively). Upon exposure to the UV radiation at 308 nm of the equimolar mixtures of thrombin with the photoaptamer construct, the equal yield of the crosslinked complexes was observed at concentrations which were lower by two orders of magnitude than in the case of the primary photoaptamer. It was found that concurrently with crosslinking to thrombin a photo-induced inactivation of the photoaptamer occurs presumably due to formation of the intermolecular crosslinking.
Download PDF:

Rakhmetova S.Yu., Radko S.P., Gnedenko O.V., Bodoev N.V., Ivanov A.S., Archakov A.I. (2010) Biomeditsinskaya khimiya, 56(1), 72-81.
This paper is also available as the English translation:10.1134/S1990750810010099
 2019 (vol 65)
 2018 (vol 64)
 2017 (vol 63)
 2016 (vol 62)
 2015 (vol 61)
 2014 (vol 60)
 2013 (vol 59)
 2012 (vol 58)
 2011 (vol 57)
 2010 (vol 56)
 2009 (vol 55)
 2008 (vol 54)
 2007 (vol 53)
 2006 (vol 52)
 2005 (vol 51)
 2004 (vol 50)
 2003 (vol 49)

© Institute of Biomedical Chemistry, Moscow, Russia