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.     

Alpha-L-RNA (alpha-L-ribo configured RNA): synthesis and RNA-selective hybridization of alpha-L-RNA/alpha-L-LNA chimera

Synthesis of the novel alpha-L-ribofuranosyl phosphoramidite derivative was accomplished via the alpha-L-ribofuranosyl thymine nucleoside. Amidite was used in automated syntheses of chimeric oligonucleotides composed of mixtures of the novel alpha-L-RNA nucleotide monomer ((alphaL)T, alpha-L-ribo configured RNA), and DNA, LNA (T(L), locked nucleic acid) or alpha-L-LNA ((alphaL)T(L), alpha-L-ribo configured locked nucleic acid) nucleotide monomers. For alpha-L-RNA/DNA and alpha-L-RNA/alpha-L-LNA chimeras, RNA-selective hybridization was obtained, for alpha-L-RNA/alpha-L-LNA chimera we found increased binding affinity compared to the corresponding DNA:RNA reference duplex. In addition, alpha-L-RNA/alpha-L-LNA chimera displayed significant stabilization towards 3'-exonucleolytic degradation. These results indicate that alpha-L-RNA/alpha-L-LNA chimeras deserve further evaluation as antisense molecules.     

The adenine derivative of alpha-L-LNA (alpha-L-ribo configured locked nucleic acid): synthesis and high-affinity hybridization towards DNA, RNA, LNA and alpha-L-LNA complementary sequences

Synthesis of a 9-mer alpha-L-LNA (alpha-L-ribo configured locked nucleic acid) containing three 9-(2-O,4-C-methylene-alpha-L-ribofuranosyl)adenine nucleotide monomer(s) has been accomplished. The work involved synthesis of the bicyclic adenine nucleoside via a condensation reaction between L-threo-pentofuranose derivative 1 and 6-N-benzoyladenine followed by C2'-epimerization. Hybridization studies demonstrated very strong duplex formation with 9-mer complementary DNA, RNA, LNA and alpha-L-LNA target sequences.     

Structural characterization of LNA and alpha-L-LNA hybridized to RNA

LNA and alpha-L-LNA are promising candidates for the development of efficient oligonucleotide-based therapeutic agents. Here, we present a short overview of the structural results we have obtained for LNA:RNA and alpha-L-LNA:RNA hybrids. Specifically, we have shown that LNA acts as an A-type mimic, while alpha-L-LNA acts as a B-type mimic when built into oligonucleotides.     

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 alpha-L-LNA (the alpha-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 alpha-L-LNA sequences in relation to the unprecedented binding properties of LNA and alpha-L-LNA.     

Nuclease resistant methylphosphonate-DNA/LNA chimeric oligonucleotides

Synthesis of chimeric 9-mer oligonucleotides containing methylphosphonate-linkages and locked nucleic acid (LNA) monomers, their binding affinity towards complementary DNA and RNA, and their 3′-exonucleolytic stability are described. The obtained methylphosphonate-DNA/LNA chimeric oligonucleotides display similarly high RNA affinity and RNA selectivity as a corresponding 9-mer DNA/LNA chimeric oligonucleotide, but much higher resistance towards 3′-exonucleolytic degradation.     

Expanding the design horizon of antisense oligonucleotides with alpha-L-LNA

Oligonucleotides containing Locked Nucleic Acids (LNA) to various extents and at various positions were evaluated for antisense activity, RNase H recruitment, nuclease stability and thermal affinity. In this work, two different diastereoisomers of LNA were studied: the beta-D-LNA and the alpha-L-LNA (abbreviated as beta-D-LNA and alpha-L-LNA). Our findings show that the best antisense activity with 16mer gapmers containing beta-D-LNA (oligonucleotides containing consecutive segments of LNA and DNA with a central DNA stretch flanked by two LNA segments, LNA-DNA-LNA) is found with gap sizes between 7 and 10 nt. The optimal gap size is motif-dependent, and requires the right balance between gap size and affinity. Compared to beta-D-LNA, alpha-L-LNA shows superior stability against a 3'-exonuclease. The design possibilities of alpha-L-LNA were explored for different gapmers and other designs, collectively called chimeras. The placement of alpha-L-LNA in the junctions or in the flanks resulted in potent antisense oligonucleotides. Moreover, different chimeras with an alternate composition of DNA, alpha-L-LNA and beta-D-LNA were evaluated in terms of antisense activity and RNase H recruitment. Chimeras with an interrupted DNA stretch with alpha-L-LNA still recruit RNase H and show good levels of antisense activity, while the same design with beta-D-LNA results in a drop in antisense potency. Our findings indicate that alpha-L-LNA is a powerful and versatile nucleotide analogue for designing potent antisense oligonucleotides.     

Aptamers targeted to an RNA hairpin show improved specificity compared to that of complementary oligonucleotides

Aptamers interacting with RNA hairpins through loop-loop (so-called kissing) interactions have been described as an alternative to antisense oligomers for the recognition of RNA hairpins. R06, an RNA aptamer, was previously shown to form a kissing complex with the TAR (trans-activating responsive) hairpin of HIV-1 RNA (Ducongé and Toulmé (1999) RNA 5, 1605). We derived a chimeric locked nucleic acid (LNA)/DNA aptamer from R06 that retains the binding properties of the originally selected R06 aptamer. We demonstrated that this LNA/DNA aptamer competes with a peptide of the retroviral protein Tat for binding to TAR, even though the binding sites of the two ligands do not overlap each other. This suggests that upon binding, the aptamer TAR adopts a conformation that is no longer appropriate for Tat association. In contrast, a LNA/DNA antisense oligomer, which exhibits the same binding constant and displays the same base-pairing potential as the chimeric aptamer, does not compete with Tat. Moreover, we showed that the LNA/DNA aptamer is a more specific TAR binder than the LNA/DNA antisense sequence. These results demonstrate the benefit of reading the three-dimensional shape of an RNA target rather than its primary sequence for the design of highly specific oligonucleotides.     

Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates

The potency and specificity of locked nucleic acid (LNA) antisense oligonucleotides was investigated as a function of length and affinity. The oligonucleotides were designed to target apolipoprotein B (apoB) and were investigated both in vitro and in vivo. The high affinity of LNA enabled the design of short antisense oligonucleotides (12- to 13-mers) that possessed high affinity and increased potency both in vitro and in vivo compared to longer oligonucleotides. The short LNA oligonucleotides were more target specific, and they exhibited the same biodistribution and tissue half-life as longer oligonucleotides. Pharmacology studies in both mice and non-human primates were conducted with a 13-mer LNA oligonucleotide against apoB, and the data showed that repeated dosing of the 13-mer at 1–2 mg/kg/week was sufficient to provide a significant and long lasting lowering of non-high-density lipoprotein (non-HDL) cholesterol without increasing serum liver toxicity markers. The data presented here show that oligonucleotide length as a parameter needs to be considered in the design of antisense oligonucleotide and that potent short oligonucleotides with sufficient target affinity can be generated using the LNA chemistry. Conclusively, we present a 13-mer LNA oligonucleotide with therapeutic potential that produce beneficial cholesterol lowering effect in non-human primates.     

Nuclease stability of LNA oligonucleotides and LNA-DNA chimeras

The stabilizing properties of LNA and alpha-L-LNA oligonucleotides against endo- and 3'-exonucleases have been evaluated.     

Synthesis, nucleic acid hybridization properties and molecular modelling studies of conformationally restricted 3'-O,4'-C-methylene-linked alpha-L-ribonucleotides

Nucleotides with conformationally restricted carbohydrate rings such as locked nucleic acid (LNA), alpha-L-LNA or 2',5'-linked 3'-O,4'-C-methyleribonucleotides exhibit significant potential as building blocks for antigene and antisense strategies. 2',5'-Linked alpha-L-ribo configured monomer X (termed alpha-L-ONA) was designed as a potential structural mimic of alpha-L-LNA. The corresponding phosphoramidite building block of monomer X was obtained in five steps (10% overall yield) from the easily obtainable thymine derivative 1. Incorporation of monomer X into oligodeoxyribonucleotides (ONs) results in dramatically decreased thermal stabilities with DNA/RNA complements (DeltaTm/mod=-11.5 to -17.0 degrees C) compared to unmodified reference ONs. Less pronounced decreases (DeltaTm/mod=-4.5 to -8.5 degrees C) are observed when monomer X is incorporated into triplex forming ONs and targeted against double-stranded DNA (parallel orientation, pyrimidine motif). This biophysical data, together with modelling studies, suggest that 2',5'-linked alpha-L-ONA is a poor structural mimic of alpha-L-LNA.     

Structure and polymorphism of the UL6 portal protein of herpes simplex virus type 1

By electron microscopy and image analysis, we find that baculovirus-expressed UL6 is polymorphic, consisting of rings of 11-, 12-, 13-, and 14-fold symmetry. The 12-mer is likely to be the oligomer incorporated into procapsids: at a resolution of 16 A, it has an axial channel, peripheral flanges, and fits snugly into a vacant vertex site. Its architecture resembles those of bacteriophage portal/connector proteins.     

Methylphosphonate LNA: a locked nucleic acid with a methylphosphonate linkage

Synthesis of an oligonucleotide containing one methylphosphonate locked nucleic acid (LNA) thymine monomer using the phosphoramidite approach is described. The binding affinity of this 9-mer methylphosphonate LNA towards complementary DNA and RNA oligonucleotides was increased compared to the reference DNA, but decreased compared to the reference LNA. In the 9-mer sequence context studied, introduction of a single methylphosphonate LNA monomer, contrary to a single LNA monomer, efficiently inhibits 3'-exonucleolytic degradation.     

Antisense inhibition of RNase P: mechanistic aspects and application to live bacteria

We explored bacterial RNase P as a drug target using antisense oligomers against the P15 loop region of Escherichia coli RNase P RNA. An RNA 14-mer, or locked nucleic acid (LNA) and peptide nucleic acid (PNA) versions thereof, disrupted local secondary structure in the catalytic core, forming hybrid duplexes over their entire length. Binding of the PNA and LNA 14-mers to RNase P RNA in vitro was essentially irreversible and even resisted denaturing PAGE. Association rates for the RNA, LNA, and PNA 14-mers were approximately 10(5) m(-1) s(-1) with a rate advantage for PNA and were thus rather fast despite the need to disrupt local structure. Conjugates in which the PNA 14-mer was coupled to an invasive peptide via a novel monoglycine linker showed RNase P RNA-specific growth inhibition of E. coli cells. Cell growth could be rescued when expressing a second bacterial RNase P RNA with an unrelated sequence in the target region. We report here for the first time specific and growth-inhibitory drug targeting of RNase P in live bacteria. This is also the first example of a duplex-forming oligomer that invades a structured catalytic RNA and inactivates the RNA by (i) trapping it in a state in which the catalytic core is partially unfolded, (ii) sterically interfering with substrate binding, and (iii) perturbing the coordination of catalytically relevant Mg2+ ions.     

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.     

Bent oligonucleotide duplexes as HMGB1 inhibitors: a comparative study

In this work we explore the ability of a chimeric LNA/DNA bent duplex, in which the kink is induced by 2 unpaired adenines in the middle of one strand, to bind HMGB1, a protein involved in many inflammatory processes. The LNA/DNA duplex was compared with the corresponding full DNA and PNA/DNA chimera duplexes from a thermodynamic and spectroscopic point of view.