The affinity of an 8-bromodeoxyguanosine- (8-BrdG-) substituted thrombin-binding aptamer (TBA-Br), which has the 1st and 10th guanosine residues replaced with 8-BrdG, was estimated using reflectometric interference spectroscopy (RIfS). When comparing TBA-Br with unmodified TBA (TBA-H), it was demonstrated that the modification effectively improved the affinity of TBA; dissociation constants ( ) of TBA-H and TBA-Br were 45.4？nM and 1.99？nM, respectively. These values, which were obtained by direct observation of thrombin binding using RIfS, have the same order of magnitude as those obtained in our previous study utilizing conformational changes in TBA to detect thrombin binding, thus confirming the validity of the obtained values. RIfS measurements also revealed that the 8-BrdG modification resulted in a lower dissociation rate constant ( ), which suggests that the enhancement of affinity can be attributed to the stabilization of the G-quadruplex structure on introduction of 8-BrdG. 1. Introduction Chemical modification of nucleic acids is a useful approach to improve the stability of higher-order structures [1–5]. Thrombin-binding aptamer (TBA), d(GGTTGGTGTGGTTGG), is a nucleic acid for which modification has attracted considerable attention, as the improved stability may lead to enhanced affinity for the target species, thrombin, which is potentially useful for the diagnosis and treatment of various conditions. Because the relationship between higher-order structure and affinity is well known, its modification has been widely studied. TBA is known to fold into a G-quadruplex structure consisting of two G-planes connected via a TGT loop and two TT loops, as shown in Figure 1(a) [6–11]. Figure 1: Structure of (a) G-quadruplex of thrombin binding aptamer and (b) 8-bromodeoxyguanosine. Modification sites, G1 and G10, are indicated in red. Strategies for modifying TBA can be classified into two categories: modification of the nucleotide backbone such as Locked Nucleic Acid (LNA) [12, 13] or 2′-fluoro-arabinonucleic acid (2′-F-ANA) [14] and modification of nucleobases, such as alkylation or phenylation [15]. Both strategies have been shown to be effective for stabilizing higher-order structure by stabilizing specific glycosidic bond conformations in the G-plane. We recently demonstrated that the higher-order structure of TBA can be stabilized by introducing 8-bromodeoxyguanosine (8-BrdG, Figure 1(b)), which stabilizes a syn conformation of a glycosidic bond by steric hindrance between the bromo group at the C8 position and the deoxyribose moiety [16–18].
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