A family of DNA aptamers with varied duplex region length that forms complexes with thrombin and prothrombin

Abstract

Edited by Judit OvádiKeywords: Aptamer Thrombin Prothrombin Surface plasmon resonance Equilibrium constant Kinetic constant a b s t r a c t Structural properties determine binding affinities of DNA aptamers specific to thrombin. Our paper is the first to focus on a family of eight G-quadruplex-based aptamers with varied duplex region length (from two to eight base pairs). We have shown that the duplex, which is not the main binding domain, greatly influences the interaction with thrombin and prothrombin. Furthermore, the affinity of an aptamer to thrombin and prothrombin increases (respectively from 2.7 Â 10 À8 M to 5.6 Â 10 À10 M and from 1.8 Â 10 À5 M to 7.1 Â 10 À9 M) with an increase in the number of nucleotide pairs in the duplex region.

Different duplex/quadruplex junctions determine the properties of anti-thrombin aptamers with mixed folding

“…In the present crystal, the duplex of RE31 establishes contacts with three different regions of the protein surface of symmetry related thrombin molecules. Although these contacts involve a smaller number of interactions with respect to the aptamer/protein binding site described above, they may contribute to the greater protein affinity measured for RE31 with respect to TBA ( 27 ). On the other hand, the compact structure of the aptamer, and in particular the recruitment of the TGT loop in the junction, may increase the resistance to nucleases that primarily attack single-stranded oligonucleotide fragments (quadruplex loops) ( 45 , 46 ) and explain the greater inhibitory effect of RE31 compared to that provided by TBA.…”

“…HD22 aptamers ( 24 ) include a 27- and a 29-mer able to bind the exosite II of thrombin with an affinity about 100–200 times higher than that of the best known thrombin binding aptamer TBA, which recognizes exosite I ( 19 ). The 31-mer RE31 ( 25 ) is a new generation aptamer with increased affinity towards exosite I ( 26 , 27 ) and improved performances in biological fluids ( 28 ) with respect to TBA. The latter has been shown ( 29 – 32 ) to adopt a unimolecular G-quadruplex structure that includes two G-tetrads, two TT loops and a TGT loop (Figure 1A ).…”

Improved RE31 Analogues Containing Modified Nucleic Acid Monomers: Thermodynamic, Structural, and Biological Effects

“…It was previously reported that the CD spectrum for RE31 displays a shape, which is characteristic for antiparallel folding topology with two positive CD bands near 245 and 295 nm and one negative signal around 265 nm. 17,34 The CD spectra for oligomers O1-O13 containing one or more nucleoside residues in UNA, LNA and β-L-RNA series were recorded to determine the influence of certain modifications on aptamer conformation. All RE31 variants displayed the typical pattern for an antiparallel G-quadruplex structure with two maxima near 245 and 295 nm and one minimum around 265 nm (Figure 2A and 2B).…”

Structure, properties, and biological relevance of the DNA and RNA G-quadruplexes: Overview 50 years after their discovery

“…In other types of duplex-G4 hybrids, complementa ry sequences were attached to the 5′ and 3′ ends of a G4 motif [189,190]. It was shown using the thrombin bind ing aptamer as an example that both duplex and antipar allel G quadruplex domains coexist in intramolecular structure of aptamer.…”

“…Despite intensive investigation of protein-G4 com plexes, the molecular mechanisms for recognition of G quadruplexes by protein molecules remain poorly under stood, and investigations in this area are fragmentary and unsystematic. It was established using high resolution methods that protein-nucleic acid binding is affected by the G4 loops [112] and duplex-G quadruplex junction [189,285]. It was shown by molecular modeling that the cavities in the HIV 1 integrase and RecA protein matched exactly the G4 size [55], and the domain of the nucleophosmin fit the G4 groove [286].…”