## 3 Unlocked Nucleic Acids (UNA)
It was found that an aptamer analog carrying UNA-U at position 7 (a loop position) inhibited blood clotting more than the unmodified compound. This research was continued with the UNA-2\({}^{\prime}\)-C-(piperazin-N-yl)-U moiety, in which the piperazine unit is protonated under physiological conditions [17]. This modification was chosen due to reports in the literature that this derivative destabilizes DNA and DNA/RNA duplexes less than regular UNA monomers [10]. A single 2\({}^{\prime}\)-C-(piperazin-N-yl)-UNA residue or 2-3 UNA residues were incorporated at positions 3, 7 or 12 (_vide supra_) of the aptamer and the thermodynamics, kinetics and biological properties were evaluated. It was found that the UNA unit with the piperazine tag provided more efficient stabilization of the quadruplex compared to regular UNA (by 0.28-0.44 kcal/mol) in a position-dependent manner, with retained quadruplex topology and molecularity. However, this generally had a negative effect on binding affinity and biological activity, with the exception of those at position 7, as the binding affinity and inhibitory properties were comparable to those of the unmodified aptamer. Finally, a UNA moiety modified within a nucleobase was tested, namely a 4-thiouracil (\({}^{\text{54}}\)U) derivative [18]. As in the previous experiments, the most favorable thermodynamic parameters were obtained by introducing the UNA-\({}^{\text{54}}\)U nucleotide at positions 3, 7 or 12, although similar effects were observed for the RNA-\({}^{\text{54}}\)U unit. Introduction of UNA-3\({}^{\prime}\)-deoxy-3\({}^{\prime}\)-amino-U at the above-mentioned key positions 3, 7 or 12 (the phosphate group was attached at the 2\({}^{\prime}\)-O oxygen atom) decreased the thermal denaturation temperature of the tetraplex by 4-7 \({}^{\circ}\)C and reduced thrombin clotting times by 32-42 s, compared with the unmodified aptamer (87 s) [19]. Considering a literature report showing that 2\({}^{\prime}\)-deoxy-isoguanosine at positions 1, 8 or 10 in the thrombin-binding aptamer enhances the aptamer-thrombin interaction [20], [PERSON] and co-workers prepared 14 aptamer analogs carrying the RNAor UNA-type isoguanine derivative, but all proved to be poor anticoagulants compared to the unmodified aptamer [21]. [PERSON] and co-workers also studied an RE31 aptamer, which is a longer version of the thrombin-binding aptamer 5\({}^{\prime}\)-GCTTGGTGGTG-3\({}^{\prime}\) mentioned above. RE31 is a 31-nt oligomer (originally developed by [PERSON] and co-workers [22]) consisting of the G-quadruplex and a duplex domain linked by four nucleotides. The 15-nt G-quadruplex parts are identical in both aptamers, whereas the duplex domain of RE31 consists of six pairs of complementary nucleotides. Of the 13 analogs synthesized, 6 oligomers contained a single UNA unit (A, C, G and U), one oligomer had 3 UNA nucleotides, other compounds contained one UNA and multiple LNA nucleotides and one oligomer was modified with 14 \(\beta\)-L-RNA units [23]. The greatest increase in thermal stability of RE31 was observed for oligomers labeled with the LNA units present in the duplex fragment or with a combination of LNAs in the helix and UNAs at position 15. Additionally, ternary substitution of T11, T15 and T20 by UNA-U resulted in a 5.4 \({}^{\circ}\)C increase in Tm. Only a minor stabilizing effect was observed for the other aptamers. Structural studies confirmed that the overall shape of RE31 analogs remained unchanged and that all analyzed aptamers formed antiparallel G-quadruplexes at 37 \({}^{\circ}\)C. All analyzed analogs of RE31 possessed prolonged biological stability in human serum. Several papers have been published on the use of UNA nucleotides to improve the potency of siRNA oligomers. Typically, siRNA oligomers are RNA duplexes of 21 base pairs containing a 19-mer base-pairing stem and 2-nt 3\({}^{\prime}\)-overhangs. The discovery of their ability to knock down gene function has made significant progress in mammalian cell culture studies.