## 3 Unlocked Nucleic Acids (UNA)
However, unmodified siRNAs suffer from insufficient selectivity of the gene regulatory process and limited stability in body fluids. Therefore, the use of UNA nucleotides seemed to be at least a partial solution to these problems. Indeed, some siRNAs with UNA units proved to be remarkably stable (e.g., in mice) and, in contrast to unmodified siRNAs, they efficiently induced knock-down of eGFP expression in a pancreas tumor xenograft model [24]. However, the great complexity of the RNAi mechanism confronts researchers with many factors that must be considered to properly identify the effects of UNA-nucleotides and other chemical modifications on siRNA activity [25; 26; 27; 28]. UNA-nucleotides have also been tested as fluorescently labeled compounds to be used as sensitive probes in molecular biology or nanotechnology. Two pyrene-modified UNA monomers were synthesized and incorporated into 21-mer DNA oligonucleotides [29]. Pyrene is a widely used chromophore due to its long fluorescence lifetime, high sensitivity and ability to stabilize DNA duplexes. The reported units differed in a linker between the 2\({}^{\prime}\)-(piperazine-N-yl) moiety and the pyrene molecule, which was either an amide (**12**, X=O, Chart 3) or an alkylamine (**12**, X=H,H). It was found that both pyrene-UNA-U units increased the duplex stability compared to UNA monomers, although the corresponding unmodified reference duplexes were still the most stable. Thermodynamic studies showed that they were able to discriminate mismatches well. Pyrene excimer emission was observed in single-stranded oligonucleotides containing three pyrene-UNA-U units, which disappeared after hybridization with DNA. Interestingly, for the first time, the fluorescence intensities of oligomers containing acyclic pyrene-nucleotides were found to increase upon hybridization with DNA or RNA templates. The unit containing the alkylamino linker (**12**, X=H,H) was used to construct a quencher-free molecular beacon that showed an increase in pyrene excimer emission of more than 900% upon hybridization of a fully complementary RNA target. This probe enabled the detection of RNA in H1299 cells [30]. Two additional pyrene-labeled UNA-U were synthesized using 5\({}^{\prime}\)-O-DMT-5-iodo-uridine (**13**, Scheme 3), which was converted to regioisomeric UNA-5-(pyren-1-yl)-U (**14** and **15**) after cleavage of the C2\({}^{\prime}\)-C3\({}^{\prime}\) bond. These compounds were converted into corresponding 3\({}^{\prime}\)-O- and 2\({}^{\prime}\)-O-phosphoramide monomers suitable for the formation of 3\({}^{\prime}\)-5\({}^{\prime}\)- and 2\({}^{\prime}\)-5\({}^{\prime}\)-inter-nucleotide bonds, respectively [31]. Both units were found to destabilize _i_-motif structures at pH 5.2. Moreover, their presence in a DNA strand resulted in reduced thermal stability of duplexes formed with DNA and RNA strands, although to a lesser extent than with unmodified UNA monomers. In addition, a bis-pyrene-labeled UNA-U was synthesized (**16**, Chart 4) with one pyrene moiety attached to position 5 of the uracil and the other to the 2\({}^{\prime}\)-(piperazin-N-yl) fragment [32]. As with the mono-labeled compounds, the compound with two labels was found to decrease the thermal stability of both DNA/DNA and DNA/RNA duplexes, but the destabilization was less than in the case of the unmodified UNA monomers. This compound also exhibits high mismatch discrimination. The UV/Vis absorption and molecular modeling studies allowed to conclude that the pyrene units are localized in the major groove of a duplex and do not intercalate with the nucleobases. In the synthesis of a UNA-3\({}^{\prime}\)-O-amino-U nucleoside (**17**, Scheme 4; Note: **17** is not a 3\({}^{\prime}\)-deoxy-3\({}^{\prime}\)-amino compound), the previously developed synthon **10a** (B=Ura) was used [33].