Solution Structure of an LNA Hybridized to DNA:  NMR Study of the d(CT^LGCT^LT^LCT^LGC):d(GCAGAAGCAG) Duplex Containing Four Locked Nucleotides

Abstract

We have used two-dimensional (1)H NMR spectroscopy at 750 MHz to determine a high-resolution solution structure of an oligonucleotide containing restricted nucleotides with a 2'-O, 4'-C-methylene bridge (LNA) hybridized to the complementary DNA strand. The LNA:DNA duplex examined contained four thymidine LNA modifications (T(L), d(C1T(L)2G3C4T(L)5T(L)6C7T(L)8G9C10):d( G11C12A13G14A15A16G17C 18A19G20). A total relaxation matrix approach was used to obtain interproton distance bounds from NOESY cross-peak intensities. These distance bounds were used as restraints in molecular dynamics (rMD) calculations. Forty final structures were generated for the duplex from A-form and B-form DNA starting structures. The root-mean-square deviation (RMSD) of the coordinates for the 40 structures of the complex was 0.6 A. The sugar puckerings are averaged values of a dynamic interchange between N- and S-type conformation except in case of the locked nucleotides that were found to be fixed in the C3'-endo conformation. Among the other nucleotides in the modified strand, the furanose ring of C7 and G9 is predominantly in the N-type conformation whereas that of G3 is in a mixed conformation. The furanose rings of the nucleotides in the unmodified complementary strand are almost exclusively in the S-type conformation. Due to these different conformations of the sugars in the two strands, there is a structural strain between the A-type modified strand and the B-type unmodified complementary strand. This strain is relaxed by decreasing the value of rise and compensating with tip, buckle, and propeller twist. The values of twist vary along the strand but for a majority of the base pairs a value even lower than that of A-DNA is observed. The average twist over the sequence is 32+/-1 degrees. On the basis of the structure, we conclude that the high stability of LNA:DNA duplexes is caused by a local change of the phosphate backbone geometry that favors a higher degree of stacking.

Quantum Mechanical Studies of DNA and LNA

“…From the structural representation, DNA is generally more bent while LNA is more stretched. This is consistent with the more rigid bicyclic structure of LNA (Obika et al, 1997;Nielsen et al, 2000;Petersen et al, 2000Petersen et al, , 2002Nielsen and Spielmann, 2005;Hughesmann et al, 2011). When the iso-potential surfaces are added, the surface of LNA-PO-AAG is constricted and concentrated on the phosphate backbone face of the molecule.…”

Locked Nucleic Acids: Properties, Applications, and Perspectives

“…A strong tendency to adopt N-type conformation was found [45], which indicates a strong conformational steering by the LNA nucleotides on their more flexible 2 0 -deoxynucleotide neighbors, especially in the 3 0 -direction. Full structural studies of the above and other LNA sequences in duplexes formed with complementary DNA have shown that the B-type character of a dsDNA duplex decreases by the incorporation of LNA monomers [46,47]. Thus, the 2 0 -deoxynucleotides in an LNA-sequence are more or less forced into adapting much larger populations of N-type sugar puckering, whereas the nucleotides of the complementary DNA-sequence display only a slight increase in the population of N-type sugars.…”

“…In other words, LNA is a true RNA-mimic. The conformational changes in an LNA-sequence have been associated with an increased stacking of the nucleobases [45][46][47].…”

Chemistry Can Make Strict and Fuzzy Controls for Bio-Systems: DNA Nanoarchitectonics and Cell-Macromolecular Nanoarchitectonics

“…165,166 The stronger affinity is primarily ascribed to the "locking" of conformation of the ribose ring by the bicyclic skeleton. 167 In solutions, (unmodified) deoxyribose rings in single-stranded DNA are rather flexible and take dynamically both S-and Nconformations. When DNA forms a duplex with DNA or RNA, the conformation of deoxyribose ring must be "frozen" into Nlike conformation, and this process is accompanied by unfavorable entropy loss.…”