The Regioselective Conjugation of the 15-nt Thrombin Aptamer with an Optimized Tripeptide Sequence Greatly Increases the Anticoagulant Activity of the Aptamer

Abstract

Currently, oligonucleotide therapy has emerged as a new paradigm in the treatment of human diseases. In many cases, however, therapeutic oligonucleotides cannot be used directly without modification. Chemical modification or the conjugation of therapeutic oligonucleotides is required to increase their stability or specificity, improve their affinity or inhibitory characteristics, and address delivery issues. Recently, we proposed a conjugation strategy for a 15-nt G-quadruplex thrombin aptamer aimed at extending the recognition interface of the aptamer. In particular, we have prepared a series of designer peptide conjugates of the thrombin aptamer, showing improved anticoagulant activity. Herein, we report a new series of aptamer–peptide conjugates with optimized peptide sequences. The anti-thrombotic activity of aptamer conjugates was notably improved. The lead conjugate, TBA–GLE, was able to inhibit thrombin-induced coagulation approximately six-fold more efficiently than the unmodified aptamer. In terms of its anticoagulant activity, the TBA–GLE conjugate approaches NU172, one of the most potent G-quadruplex thrombin aptamers. Molecular dynamics studies have confirmed that the principles applied to the design of the peptide side chain are efficient instruments for improving aptamer characteristics for the proposed TBA conjugate model.

Alpha-Deoxyguanosine to Reshape the Alpha-Thrombin Binding Aptamer

“…The relative t 1/2 values referred to TBA were 2.9 ± 0.6, 5.9 ± 0.9, and 4.1 ± 0.8 for 2SLE, 2GLE, and 4GLE, respectively. Interestingly, the enhancement effect induced by the SLE and GLE peptides in analogue 2 was almost identical in magnitude to that of the previously reported conjugates TBA-SLE and TBA-GLE [20]. In the case of variant 4, the effect of the GLE subunit was apparently weakened by distortions at the aptamer-protein interface due to the presence of two modified tetrads Q3 and Q4 in the quadruplex core.…”

“…In the present study, we take advantage of the anti-preferred configuration of alphadeoxyguanosine and develop new non-natural three-and four-layer TBA analogues that form stable anti-parallel G-quadruplex structures and retain their anticoagulant activity. Furthermore, we show that the antithrombotic activity of modified G-quadruplexes can be considerably increased by regioselective conjugation with optimized tripeptide sequences, as recently described [20].…”

“…We examined the anticoagulant activity of new TBA analogues in fibrinogen clotting time tests. Similar to our previous reports [20,22], we used an increase in absorbance at 360 nm due to light scattering during alpha-thrombin-induced fibrinogen polymerization in the presence of an aptamer. The anticoagulant effect was estimated as the time required to reach 50% of the absorbance maximum.…”

“…Although most of the studied modified TBA analogues are characterized by very high thermodynamic stability and, presumably, enhanced nuclease resistance due to the presence of non-natural nucleotides, we observed some decline in their anticoagulant activity. Fortunately, in our recent study, we proposed an efficient approach to enhance the anticoagulant activity of TBA [20]. Previously, we have shown that a regioselective conjugation of an optimized tripeptide with a T3 residue at its N3 position greatly increases the anticoagulant activity of the aptamer.…”

“…Fluorescent aptamers were prepared by click reaction using propargyl-modified oligonucleotides and Sulfo-Cy5 azide (Lumiprobe). Peptide conjugates 2SLE, 2GLE, and 4GLE were prepared as described previously [20]. Purification of TBA analogues, their peptide conjugates, and labeled oligonucleotides was carried out by a combination of reversed-phase HPLC and denaturing gel electrophoresis with control by MALDI mass spectrometry.…”