## 3 Unlocked Nucleic Acids (UNA)
However, UNA nucleotides located close to the center of the oligomer make the corresponding RNA duplex structure 4.0-6.6 kcal/mol less favorable. Much smaller destabilization (0.5-1.5 kcal/mol) is caused by UNA residues located near the 5\({}^{\prime}\) or 3\({}^{\prime}\) ends. Thermodynamic analysis of a large set of UNA-modified oligomers showed that UNA pyrimidine nucleotides cause greater destabilization than purine nucleotides, and the thermodynamic effects of the UNA substitutions are additive. It was also documented that UNA nucleotides interact according to the Watson-Crick base-pairing rules, while the stacking interactions of UNA nucleotides are less favorable than those of RNA units. The UNA nucleotides have also been studied in terms of their effects on the stability of other high-ordered structures, i.e., _i_-motif and G-quadruplex. It is known that C-rich oligonucleotides can adopt _i_-motif structures, in which the rigid core is formed due to intercalation of C-C\({}^{+}\) base pairs present in a single strand or in a complex of up to four strands, possibly arranged in a few orientations [13]. Such fragments have been found in biologically important structures and appear to be important in the regulation of biological processes. A widely known _i_-motif sequence is a 22-nt fragment of human telomeric DNA with the sequence 5\({}^{\prime}\)-d(CCCTAA)\({}_{3}\)CCCT). This molecule was engineered to form twelve 22-nt oligomers, each containing a single UNA modification located in oligomers in a \"walking mode\" [14]. The thermodynamic stability of the _i_-motif structures was measured in buffers of pH 4.8 and 5.6. It was found that the UNA units, depending on their position, either destabilize (positions C1 or C14) or stabilize (U10, A11, A12) the structure. The recorded CD spectra allow to conclude that the UNA-nucleotides do not cause significant changes in the _i_-motif structure and folding molecularity. The G-quadruplexes are formed from G-rich nucleic acid fragments and their characteristic feature is a stack of planar G-quartets interacting according to the Hoogsteen scheme of hydrogen bonding. The quartets can be formed by guanosine nucleotides present in a single strand or in a complex of up to four strands, and are stabilized by several cations, in particular by potassium and sodium ions. Generally, K\({}^{+}\) cations stabilize guanine-quartet assemblies to a larger extent than Na\({}^{+}\). It is well-documented that G-quadruplexes are capable of tuning many biological processes. The influence of UNA nucleotides on the structure and stability of G-quadruplexes was investigated using CD spectroscopy, as G-quadruplexes generate unique CD signals. After analyzing a series of oligomers of the general sequence GGGTTTTTGGGGA(T,C)GGGTTTTGGG, it was found that UNA nucleotides present in the loops cause global stabilization of the structure, while those in the stem cause destabilization of the quadruplexes [15]. These observations were relevant to the study of the UNA-modified thrombin-binding aptamer 5\({}^{\prime}\)-GGTTGGTTGG-3\({}^{\prime}\), which forms an intramolecular antiparallel G-quadruplex that interacts with two thrombin molecules and inactivates only one of them. Again, a library of fifteen modified oligomers containing UNA nucleotides (distributed in a \"walking mode\") was prepared, and UV melting analysis was applied to obtain thermodynamic parameters as well as information on the stoichiometry of the folding process [16]. These studies revealed remarkable destabilization of the quadruplexes when UNA-G nucleotides were located in the segments forming the G-quadruplex structure. Substitutions of UNA-U for U were much less disruptive. Considering the \(\Delta\)G\({}^{\circ}\)\({}_{37}\) for the quadruplex formation, the UNA-U units introduced at positions 3, 7 or 12 make quadruplex folding more energetically favorable than when observed with the unmodified aptamer. The inhibitory effect of the aptamer analogs on blood clotting time was measured using a standard thrombin time assay.