## 5 Theroninol Nucleic Acids (TNA)  
The nucleoside precursors of acyclic threonineol nucleic acid (D-aTNA, **28**, Chart 9, a dinucleotide is shown) were prepared using DMT-protected D-threonineol (**29**, Scheme 6), except for the thymine derivative, for which unprotected D-threonineol **30** was used [57]. The amino groups were used to attach N1-carboxymethyl derivatives of the nucleobases C\({}^{\text{Bz}}\) or T, or N9-carboxymethyl derivatives of A\({}^{\text{Bz}}\) or G\({}^{\text{SiBu}}\) under conditions known from peptide chemistry. The product containing a thymine nucleobase was triylated and all four DMT-protected nucleosides **31** were routinely phosphorylated to yield the corresponding D-aTNA monomers **32**. Using the monomers **32**, seven oligomers (each 8-nt long) were synthesized on an automated DNA synthesizer. The coupling efficiency of the TNA monomers was as high as that of the conventional monomers, as judged from the Vis absorption of the released DMT\({}^{+}\) cation. After the recommended workup, the oligomers were purified by RP-HPLC and characterized by MALDI-TOF MS. Melting experiments showed that the D-aTNA oligomers 1\({}^{\prime}\)-GCATCAGT-3\({}^{\prime}\) and 3\({}^{\prime}\)-CGTAGTCA-1\({}^{\prime}\) formed an unexpectedly stable antiparallel duplex with Tm = 63 \({}^{\circ}\)C, compared with analogous DNA and RNA duplexes with 29 and 39 \({}^{\circ}\)C, respectively. Notably, a single T\(\circ\)C mismatch significantly lowered the Tm to 49 \({}^{\circ}\)C. CD studies showed that single-stranded D-aTNA oligomers, unlike GNA, do not form a helical preorganized structure. The same group reported the synthesis of L-aTNA using an optimized method analogous to that used in the synthesis of D-aTNA [58]. As expected from the enantiomeric relationship between L- and D-aTNA, the L-aTNAs formed an antiparallel homo-duplex with the same melting temperature (58 \({}^{\circ}\)C) as the D-aTNA duplex of the same sequence. However, unlike D-aTNA, L-aTNA not only hybridized quite well with both DNA (Tm = 25-28 \({}^{\circ}\)C) and RNA (Tm = 38-41 \({}^{\circ}\)C), but the association also occurred in a parallel manner. The crystal structure of an L-aTNA/RNA duplex (3\({}^{\prime}\)-GCAGCAGC-1\({}^{\prime}\))/(5\({}^{\prime}\)-GCUGCU\({}^{\text{Bz}}\)GC-3\({}^{\prime}\)) clearly shows right-handed helicity and a Watson-Crick base-pairing [59]. The N-type sugar puckering was found in the RNA strand of the duplex, which is usually observed in an A-type duplex. While a CD experiment identified a single-stranded L-aTNA oligomer 5\({}^{\prime}\)-  
showed a preorganized structure (CD-positive signals at 267 and 218 nm and negative bands at 247 and 203 nm) [60]. This observation was confirmed by a UV melting curve. It was also found that the homopyrimidine L-aTNA with the sequence 5\({}^{\prime}\)-TTTTTTTTTTTT-4\({}^{\prime}\)-dT-3\({}^{\prime}\) forms a highly stable triplex L-aTNA/DNA/L-aTNA with 5\({}^{\prime}\)-d(GATCCAAAAAAAAAAAAAG)-3\({}^{\prime}\) (Tm = 82 \({}^{\circ}\)C). Similarly, 5\({}^{\prime}\)-TTCTCTCTCT  
taining L-aTNA-(TT) in the 3'-overhang of the antisense strand showed 30% higher gene silencing than the siRNA carrying two natural thymidine residues. Control experiments showed that HeLa cells transfected with the _scrambled_ siRNA produced luminescence similar to the untreated cells. A time-course analysis of the siRNAs over five days showed that the siRNA containing two L-threeninol units at both 3'-ends exhibited higher gene silencing activity than the native siRNA from day 3. This duplex showed exceptional stability in human serum, with ~5% of the original siRNA still intact after 8 h, whereas the native congener was completely degraded after approximately 60 min of incubation. Furthermore, the absence of cytotoxicity of the L-threeninol modification was determined by the MTT assay.