Locked Nucleic Acid (LNA) Recognition of RNA:  NMR Solution Structures of LNA:RNA Hybrids

Abstract

Locked nucleic acids (LNAs) containing one or more 2'-O,4'-C-methylene-linked bicyclic ribonucleoside monomers possess a number of the prerequisites of an effective antisense oligonucleotide, e.g. unprecedented helical thermostability when hybridized with cognate RNA and DNA. To acquire a detailed understanding of the structural features of LNA giving rise to its remarkable properties, we have conducted structural studies by use of NMR spectroscopy and now report high-resolution structures of two LNA:RNA hybrids, the LNA strands being d(5'-CTGAT(L)ATGC-3') and d(5'-CT(L)GAT(L)AT(L)GC-3'), respectively, T(L) denoting a modified LNA monomer with a thymine base, along with the unmodified DNA:RNA hybrid. In the structures, the LNA nucleotides are positioned as to partake in base stacking and Watson-Crick base pairing, and with the inclusion of LNA nucleotides, we observe a progressive change in duplex geometry toward an A-like duplex structure. As such, with the inclusion of three LNA nucleotides, the hybrid adopts an almost canonical A-type duplex geometry, and thus it appears that the number of modifications has reached a saturation level with respect to structural changes, and that further incorporations would furnish only minute changes in the duplex structure. We attempt to rationalize the conformational steering induced by the LNA nucleotides by suggesting that the change in electronic density at the brim of the minor groove, introduced by the LNA modification, is causing an alteration of the pseudorotational profile of the 3'-flanking nucleotide, thus shifting this sugar equilibrium toward N-type conformation.

Chemical modification and design of anti-miRNA oligonucleotides

“…LNAs are a bicyclic nucleic acid that tether the 2¢-O to the 4¢-C via a methylene bridge (Figure 1), effectively locking the structure into a 3¢-endo sugar conformation. 28,29 This modification provides stabilization against nucleases and also offers the greatest increase in binding affinity of any of nucleic acid modifications in common use today, adding 1.5-6 1C per modification against an RNA target. One study examined the effect that 10 LNA substitutions had on the binding affinity of ASOs upon duplex formation with an RNA target and found that the LNA modifications gave a 24 1C T m increase compared with a DNA control when a PO backbone was used (an average of +2.4 1C per modification) and 16 1C when using a PS backbone (an average of +1.6 1C per modification).…”

Emerging roles of microRNAs as molecular switches in the integrated circuit of the cancer cell

“…The development of locked nucleic acids (LNAs) will aid the therapeutic applications of miRNAs. In these analogs, the ribose ring is locked by a methylene brigde connecting the 29-O atom with the 49-C atom (Petersen et al 2002). LNAs were shown to exhibit no toxicity at dosages capable of producing antitumor effects in vivo.…”

Interaction of the Ty3 Reverse Transcriptase Thumb Subdomain with Template-Primer

“…These 2Ј-O,4Ј-Cmethylene-linked bicyclic analogs (Fig. 4A) have the property of locking the deoxyribose ring in the C-3Ј-endo configuration, thereby increasing the local organization of the phosphate backbone (45). LNA modifications were localized within the region of the primer and template predicted to be involved in contacts with the Ty3 RT thumb subdomain (positions Ϫ3 to Ϫ7, defining position Ϫ1 as the first base pair in the catalytic center) (Fig.…”