Mads D. Sørensen

Locked Nucleic Acid: A Potent Nucleic Acid Analog in Therapeutics and Biotechnology

Locked nucleic acid (LNA) is a class of nucleic acid analogs possessing very high affinity and excellent specificity toward complementary DNA and RNA, and LNA oligonucleotides have been applied as antisense molecules both in vitro and in vivo. In this review, we briefly describe the basic physiochemical properties of LNA and some of the difficulties that may be encountered when applying LNA technology. The central part of the review focuses on the use of LNA molecules in regulation of gene expression, including delivery to cells, stability, unspecific effects, toxicity, pharmacokinetics, and design of LNA oligonucleotides. The last part evaluates LNA as a diagnostic tool in genotyping.

The Eight Stereoisomers of LNA (Locked Nucleic Acid): A Remarkable Family of Strong RNA Binding Molecules

The authors evaluated the RNA binding of all eight possible stereoisomers of LNA. Synthesis of qa-l-xylo-LNAq has been accomplished and the remaining four stereoisomers, all enantiomers of the four synthesized diastereoisomers, are indirectly evaluated by hybridization studies. The remarkable binding affinities and specificities obtained for LNA and a-l-LNA, both in a fully and in a partly modified context, establish these mols. as unique nucleic acid mimics. A strong impetus for this research has been, and continues to be, the therapeutic promises of the antisense strategy. [on SciFinder (R)]

LNA (locked nucleic acid) and the diastereoisomeric alpha-L-LNA: conformational tuning and high-affinity recognition of DNA/RNA targets.

The remarkable binding properties of LNA (Locked Nucleic Acid) and α-L-LNA (the α-L-ribo configured diastereoisomer of LNA) are summarized, and hybridization results for LNA/2′-O-Me-RNA chimera and LNAs with a “dangling” nucleotide are introduced. In addition, results from NMR investigations on the furanose conformations of the individual nucleotide monomers in different duplexes are presented. All these data are discussed with focus on the importance of conformational steering of unmodified nucleotides in partly modified LNA and α-L-LNA sequences in relation to the unprecedented binding properties of LNA and α-L-LNA.

Locked nucleoside analogues expand the potential of DNAzymes to cleave structured RNA targets

BackgroundDNAzymes cleave at predetermined sequences within RNA. A prerequisite for cleavage is that the DNAzyme can gain access to its target, and thus the DNAzyme must be capable of unfolding higher-order structures that are present in the RNA substrate. However, in many cases the RNA target sequence is hidden in a region that is too tightly structured to be accessed under physiological conditions by DNAzymes.ResultsWe investigated how incorporation of LNA (locked nucleic acid) monomers into DNAzymes improves their ability to gain access and cleave at highly-structured RNA targets. The binding arms of DNAzymes were varied in length and were substituted with up to three LNA and α-L-LNA monomers (forming LNAzymes). For one DNAzyme, the overall cleavage reaction proceeded fifty times faster after incorporation of two α-L-LNA monomers per binding arm (kobs increased from 0.014 min-1 to 0.78 min-1).ConclusionThe data demonstrate how hydrolytic performance can be enhanced by design of LNAzymes, and indicate that there are optimal lengths for the binding arms and for the number of modified LNA monomers.

LNA and α-L-LNA: Towards Therapeutic Applications

Abstract LNA and α-L-LNA are promising candidates for the development of efficient oligonucleotide-based therapeutic agents. Here, we report dose-dependent inhibition of HIV-1 Tat-dependent trans activation by a 12-mer chimeric α-L-LNA/DNA oligomer. This oligomer exhibits a dose-dependency similar to that of the corresponding 12-mer chimeric LNA/2′-O-Me-RNA oligomer. In addition, we show that incorporation of α-L-LNA or LNA monomers into each of the two binding arms of a “10–23” DNAzyme markedly increases cleavage of the target RNA.

α-l-RNA (α-l-ribo Configured RNA): Synthesis and RNA-Selective Hybridization of α-l-RNA/α-l-LNA Chimera

Abstract Synthesis of the novel α- l -ribofuranosyl phosphoramidite derivative 7 was accomplished via the α- l -ribofuranosyl thymine nucleoside 4 . Amidite 7 was used in automated syntheses of chimeric oligonucleotides composed of mixtures of the novel α- l -RNA nucleotide monomer ( αL T , α- l - ribo configured RNA), and DNA, LNA ( T L , locked nucleic acid) or α- l -LNA ( αL T L , α- l - ribo configured locked nucleic acid) nucleotide monomers. For α- l -RNA/DNA and α- l -RNA/α- l -LNA chimeras, RNA-selective hybridization was obtained, for α- l -RNA/α- l -LNA chimera we found increased binding affinity compared to the corresponding DNA:RNA reference duplex. In addition, α- l -RNA/α- l -LNA chimera displayed significant stabilization towards 3′-exonucleolytic degradation. These results indicate that α- l -RNA/α- l -LNA chimeras deserve further evaluation as antisense molecules.

Oligodeoxynucleotides containing α-L-ribo configured LNA-type C-aryl nucleotides

Synthesis of 2′-O,4′-C-methylene-α-L-ribofuranosyl derivatives containing phenyl and 1-pyrenyl aglycons, i.e., novel α-L-ribo configured LNA-type C-aryl nucleosides, has been accomplished. Key synthetic steps included stereoselective Grignard reactions on tetrahydrofuran aldehyde 12, configurational inversion of the resulting alcohol 13 into alcohol 15, and concomitant Mitsonobu cyclization furnishing the desired bicyclic furanosyl skeleton with a locked conformation. The phosphoramidite derivatives 19a and 19b were used for automated synthesis of 9-mer DNA and α-L-LNA oligonucleotides containing the α-L-LNA-type C-aryl monomers αLPhL and αLPyL containing a phenyl and pyrenyl aglycon, respectively. Thermal denaturation studies showed universal base pairing behavior for the pyrenyl monomer αLPyL when incorporated into a DNA or an α-L-LNA oligonucleotide.

Parallel nucleic acid recognition by the LNA (locked nucleic acid) stereoisomers β-L-LNA and α-D-LNA; studies in the mirror image world

Two LNA (locked nucleic acid) stereoisomers (β-L-LNA and α-D-LNA) are evaluated in the mirror-image world, that is by the study of two mixed sequences of LNA and α-L-LNA and their L-DNA and L-RNA complements. Both are found to display high-affinity RNA-recognition by the formation of duplexes with parallel strand orientation.