Katrine E. Nielsen

The conformations of locked nucleic acids (LNA)

We have used 2D NMR spectroscopy to study the sugar conformations of oligonucleotides containing a conformationally restricted nucleotide (LNA) with a 2′‐O, 4′‐C‐methylene bridge. We have investigated a modified 9‐mer single stranded oligonucleotide as well as three 9‐ and 10‐mer modified oligonucleotides hybridized to unmodified DNA. The single‐stranded LNA contained three modifications whereas the duplexes contained one, three and four modifications, respectively. The LNA:DNA duplexes have normal Watson–Crick base‐pairing with all the nucleotides in anti‐conformation. By use of selective DQF‐COSY spectra we determined the ratio between the N‐type (C3′‐endo) and S‐type (C2′‐endo) sugar conformations of the nucleotides. In contrast to the corresponding single‐stranded DNA (ssDNA), we found that the sugar conformations of the single‐stranded LNA oligonucleotide (ssLNA) cannot be described by a major S‐type conformer of all the nucleotides. The nucleotides flanking an LNA nucleotide have sugar conformations with a significant population of the N‐type conformer. Similarly, the sugar conformations of the nucleotides in the LNA:DNA duplexes flanking a modification were also shown to have significant contributions from the N‐type conformation. In all cases, the sugar conformations of the nucleotides in the complementary DNA strand in the duplex remain in the S‐type conformation. We found that the locked conformation of the LNA nucleotides both in ssLNA and in the duplexes organize the phosphate backbone in such a way as to introduce higher population of the N‐type conformation. These conformational changes are associated with an improved stacking of the nucleobases. Based on the results reported herein, we propose that the exceptional stability of the LNA modified duplexes is caused by a quenching of concerted local backbone motions (preorganization) by the LNA nucleotides in ssLNA so as to decrease the entropy loss on duplex formation combined with a more efficient stacking of the nucleobases. Copyright

α‐L‐LNA (α‐L‐ribo Configured Locked Nucleic Acid) Recognition of DNA: An NMR Spectroscopic Study

We have used NMR and CD spectroscopy to study and characterise two α-L-LNA:DNA duplexes, a nonamer that incorporates three α-L-LNA nucleotides and a decamer that incorporates four α-L-LNA nucleotides, in which α-L-LNA is α-L-ribo-configured locked nucleic acid. Both duplexes adopt right-handed helical conformations and form normal Watson–Crick base pairing with all nucleobases in the anti conformation. Deoxyribose conformations were determined from measurements of scalar coupling constants in the sugar rings, and for the decamer duplex, distance information was derived from 1H–1H NOE measurements. In general, the deoxyriboses in both of the α-L-LNA:DNA duplexes adopt S-type (B-type structure) sugar puckers, that is the inclusion of the modified α-L-LNA nucleotides does not perturb the local native B-like double-stranded DNA (dsDNA) structure. The CD spectra of the duplexes confirm these findings, as these display B-type characteristic features that allow us to characterise the overall duplex type as B-like. The 1H–1H NOE distances which were determined for the decamer duplex were employed in a simulated annealing protocol to generate a model structure for this duplex, thus allowing a more detailed inspection of the impact of the α-L-ribo-configured nucleotides. In this structure, it is evident that the malleable DNA backbone rearranges in the vicinity of the modified nucleotides in order to accommodate them and present their nucleobases in a geometry suitable for Watson–Crick base pairing.

Synthesis and NMR-studies of dinucleotides with conformationally restricted cyclic phosphotriester linkages

Abstract Four diastereomeric dinucleotides in which the phosphodiester linkages are conformationally restricted in cyclic phosphotriester structures are synthesised. From the epimeric 5′- C -vinyl thymidine derivatives, dinucleotides containing two terminal alkene moieties are constructed via standard phosphoramidite chemistry, and applied as substrates in ring-closing metathesis (RCM) reactions. Hereby, four diastereomeric dinucleotides with seven membered phosphepine rings in the inter-nucleoside linkages are obtained and separated, and their configurations elucidated by advanced NMR-studies in combination with restrained molecular dynamics (rMD) simulations. The seven membered rings are found to give some degree of conformational restriction in the natural nucleic acid backbone, and one of the four dinucleotides is found to favour stacking between the two adjacent thymine moieties.

Synthesis and structural characterization of piperazino-modified DNA that favours hybridization towards DNA over RNA

We report the synthesis of two C4′-modified DNA analogues and characterize their structural impact on dsDNA duplexes. The 4′-C-piperazinomethyl modification stabilizes dsDNA by up to 5°C per incorporation. Extension of the modification with a butanoyl-linked pyrene increases the dsDNA stabilization to a maximum of 9°C per incorporation. Using fluorescence, ultraviolet and nuclear magnetic resonance (NMR) spectroscopy, we show that the stabilization is achieved by pyrene intercalation in the dsDNA duplex. The pyrene moiety is not restricted to one intercalation site but rather switches between multiple sites in intermediate exchange on the NMR timescale, resulting in broad lines in NMR spectra. We identified two intercalation sites with NOE data showing that the pyrene prefers to intercalate one base pair away from the modified nucleotide with its linker curled up in the minor groove. Both modifications are tolerated in DNA:RNA hybrids but leave their melting temperatures virtually unaffected. Fluorescence data indicate that the pyrene moiety is residing outside the helix. The available data suggest that the DNA discrimination is due to (i) the positive charge of the piperazino ring having a greater impact in the narrow and deep minor groove of a B-type dsDNA duplex than in the wide and shallow minor groove of an A-type DNA:RNA hybrid and (ii) the B-type dsDNA duplex allowing the pyrene to intercalate and bury its apolar surface.