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 β-d-LNA and α-l-LNA). Our findings show that the best antisense activity with 16mer gapmers containing β-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 β-d-LNA, α-l-LNA shows superior stability against a 3′-exonuclease. The design possibilities of α-l-LNA were explored for different gapmers and other designs, collectively called chimeras. The placement of α-l-LNA in the junctions or in the flanks resulted in potent antisense oligonucleotides. Moreover, different chimeras with an alternate composition of DNA, α-l-LNA and β-d-LNA were evaluated in terms of antisense activity and RNase H recruitment. Chimeras with an interrupted DNA stretch with α-l-LNA still recruit RNase H and show good levels of antisense activity, while the same design with β-d-LNA results in a drop in antisense potency. Our findings indicate that α-l-LNA is a powerful and versatile nucleotide analogue for designing potent antisense oligonucleotides.
Aptamers constitute a potential class of therapeutic molecules typically selected from a large pool of oligonucleotides against a specific target. With a scope of developing unique shorter aptamers with very high biostability and affinity, locked nucleic acid (LNA) nucleotides have been investigated as a substrate for various polymerases. Various reports showed that some thermophilic B-family DNA polymerases, particularly KOD and Phusion DNA polymerases, accepted LNA-nucleoside 5′-triphosphates as substrates. In this study, we investigated the docking of LNA nucleotides in the active sites of RB69 and KOD DNA polymerases by molecular docking simulations. The study revealed that the incoming LNA-TTP is bound in the active site of the RB69 and KOD DNA polymerases in a manner similar to that seen in the case of dTTP, and with LNA structure, there is no other option than the locked C3′-endo conformation which in fact helps better orienting within the active site.
Double-stranded oligonucleotides (ODNs) containing the consensus binding sequence of a transcription factor provide a rationally designed tool to manipulate gene expression at the transcriptional level by the decoy approach. However, modifications introduced into oligonucleotides to increase stability quite often do not guarantee that transcription factor affinity and/or specificity of recognition are retained. We have previously evaluated the use of locked nucleic acids (LNA) in the design of decoy molecules for the transcription factor κB. Oligo nucleotides containing LNA substitutions displayed high resistance to exo- and endonucleolytic degradation, with LNA–DNA mix-mers being more stable than LNA–DNA–LNA gap-mers. However, insertion of internal LNA bases resulted in a loss of affinity for the transcription factor. This latter effect apparently depended on positioning of the internal LNA substitutions. Indeed, here we demonstrate that intra- and inter-strand positioning of internal LNAs has to be carefully considered to maintain affinity and achieve high stability, respectively. Unfortun ately, our data also indicate that LNA positioning is not the only parameter affecting transcription factor binding, the interference in part being dependent on the intrinsic conformational properties of this nucleotide analog. To circumvent this problem, the successful use of an α-l-ribo- configured LNA is demonstrated, indicating LNA–DNA–α-l-LNA molecules as promising new decoy agents.
Modified nucleotides are increasingly being utilized in the de novo selection of aptamers for enhancing their drug-like character and abolishing the need for time consuming trial-and-error based post-selection modifications. Locked nucleic acid (LNA) is one of the most prominent and successful nucleic acid analogues because of its remarkable properties, and widely explored as building blocks in therapeutic oligonucleotides. Evolution of LNA-modified RNA aptamers requires an efficient reverse transcription method for PCR enrichment of the selected RNA aptamer candidates. Establishing this key step is a pre-requisite for performing LNA-modified RNA aptamer selection.
In this study three different reverse transcriptases were investigated towards the enzymatic recognition of LNA nucleotides. Both incorporation as well as reading capabilities of the LNA nucleotides was investigated to fully understand the limitations of the enzymatic recognition.
We found that SuperScript® III Reverse Transcriptase is an efficient enzyme for the recognition of LNA nucleotides, making it a prime candidate to be used in de novo selection of LNA containing RNA aptamers
Locked nucleic acids (LNA) are novel high-affinity DNA analogs that can be used as genotype-specific drugs. The LNA oligonucleotides (LNA PO ODNs) are very stable in vitro and in vivo without the need for a phosphorothiolated backbone. In this study we tested the biological fate and the efficacy in tumor growth inhibition of antisense oligonucleotides directed against the gene of the large subunit of RNA polymerase II (POLR2A) that are completely synthesized as LNA containing diester backbones. These full LNA oligonucleotides strongly reduce POLR2A protein levels. Full LNA PO ODNs appeared to be very stable compounds when injected into the circulation of mice. Full LNA PO ODNs were continuously administered for 14 days to tumor-bearing nude mice. Tumor growth was inhibited sequence specifically at dosages from 1 mg/kg/day. LNA PO ODNs appeared to be non-toxic at dosages <5 mg/kg/day. Biodistribution studies showed the kidneys to have the highest uptake of LNA PO ODNs and urinary secretion as the major route of clearance. This report shows that LNA PO ODNs are potent genotype-specific drugs that can inhibit tumor growth in vivo.
Locked nucleic acid (LNA) is a chemically modified nucleic acid with its sugar ring locked in an RNA-like (C3′-endo) conformation. LNAs show extraordinary thermal stabilities when hybridized with DNA, RNA or LNA itself. We performed molecular dynamics simulations on five isosequential duplexes (LNA–DNA, LNA–LNA, LNA–RNA, RNA–DNA and RNA–RNA) in order to characterize their structure, dynamics and hydration. Structurally, the LNA–DNA and LNA–RNA duplexes are found to be similar to regular RNA–DNA and RNA–RNA duplexes, whereas the LNA–LNA duplex is found to have its helix partly unwound and does not resemble RNA–RNA duplex in a number of properties. Duplexes with an LNA strand have on average longer interstrand phosphate distances compared to RNA–DNA and RNA–RNA duplexes. Furthermore, intrastrand phosphate distances in LNA strands are found to be shorter than in DNA and slightly shorter than in RNA. In case of induced sugar puckering, LNA is found to tune the sugar puckers in partner DNA strand toward C3′-endo conformations more efficiently than RNA. The LNA–LNA duplex has lesser backbone flexibility compared to the RNA–RNA duplex. Finally, LNA is less hydrated compared to DNA or RNA but is found to have a well-organized water structure.
Oligonucleotides containing locked nucleic acid bases (LNAs) have increased affinity for complementary DNA sequences. We hypothesized that enhanced affinity might allow LNAs to recognize chromosomal DNA inside human cells and inhibit gene expression. To test this hypothesis, we synthesized antigene LNAs (agLNAs) complementary to sequences within the promoters of progesterone receptor (PR) and androgen receptor (AR). We observed inhibition of AR and PR expression by agLNAs but not by analogous oligomers containing 2'-methoxyethyl bases or noncomplementary LNAs. Inhibition was dose dependent and exhibited IC50 values of <10 nM. Efficient inhibition depended on the length of the agLNA, the location of LNA bases, the number of LNA substitutions, and the location of the target sequence within the targeted promoter. LNAs targeting sequences at or near transcription start sites yielded better inhibition than LNAs targeting transcription factor binding sites or an inverted repeat. These results demonstrate that agLNAs can recognize chromosomal target sequences and efficiently block gene expression. agLNAs could be used, for gene silencing, as cellular probes for chromosome structure, and therapeutic applications.
Transfection of cis-element double-stranded oligonucleotides, referred to as decoy ODNs, has been reported to be a powerful tool that provides a new class of antigene strategies for gene therapy. However, one of the major limitations of the decoy approach is the rapid degradation of phosphodiester oligonucleotides by intracellular nucleases. To date, several DNA analogs have been employed to overcome this issue, but insufficient efficacy and/or specificity have limited their in vivo usefulness. In this paper we have investigated the use of conformationally restricted nucleotides in the design of decoy molecules for nuclear transcription factor κB (NF-κB). Starting from a synthetic double-stranded oligonucleotide, containing the κB consensus binding sequence, we designed a panel of decoy molecules modified to various extents and at various positions with locked nucleic acids (LNAs). Our results indicate that the addition of terminal LNA bases, outside the κB sequence, to generate LNA–DNA–LNA co-polymers was sufficient to confer appreciable protection towards nuclease digestion, without interfering with transcription factor binding. Conversely, insertion of LNA substitutions in the context of the κB-binding site resulted in further increased stability, but caused a loss of affinity of NF-κB for the target sequence. However, our results also indicate that this latter effect was apparently dependent not only on the extent but also on strand positioning of the internal LNA substitutions. This observation is of great importance since it provides evidence for the possibility of tuning DNA–LNA duplexes with internal LNAs into decoy agents with improved features in terms of biological stability and inhibitory effect.
The effect of locked nucleic acid (LNA) modification position upon representative DNA polymerase and exonuclease activities has been examined for potential use in primer extension genotyping applications. For the 3′→5′ exonuclease activities of four proofreading DNA polymerases (Vent, Pfu, Klenow fragment and T7 DNA polymerase) as well as exonuclease III, an LNA at the terminal (L-1) position of a primer is found to provide partial protection against the exonucleases of the two family B polymerases only. In contrast, an LNA residue at the penultimate (L-2) position generates essentially complete nuclease resistance. The polymerase active sites of these enzymes also display a distinct preference. An L-1 LNA modification has modest effects upon poly merization, but an L-2 LNA group slows dTTP incorporation somewhat while virtually abolishing extension with ddTTP or acyTTP terminators, even with A488L Vent DNA polymerase engineered for terminator incorporation. These observations on active site preference have been utilized to demonstrate two novel assays: exonuclease-mediated single base extension (E-SBE) and proofreading allele-specific extension (PRASE). We show that a model PRASE genotyping reaction with L-2 LNA primers offers greater specificity than existing non-proofreading assays, whether or not the non-proofreading reaction employs LNA-modified primers.
Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.
Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.
Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).
Immunology; Issue 59; fluorescence in situ hybridization; FISH; flow cytometry; locked nucleic acid; sRNA; Vibrio
In spite of the many developments in synthetic oligonucleotide (ON) chemistry and design, invasion into double-stranded DNA (DSI) under physiological salt and pH conditions remains a challenge. In this work, we provide a new ON tool based on locked nucleic acids (LNAs), designed for strand invasion into duplex DNA (DSI). We thus report on the development of a clamp type of LNA ON—bisLNA—with capacity to bind and invade into supercoiled double-stranded DNA. The bisLNA links a triplex-forming, Hoogsteen-binding, targeting arm with a strand-invading Watson–Crick binding arm. Optimization was carried out by varying the number and location of LNA nucleotides and the length of the triplex-forming versus strand-invading arms. Single-strand regions in target duplex DNA were mapped using chemical probing. By combining design and increase in LNA content, it was possible to achieve a 100-fold increase in potency with 30% DSI at 450 nM using a bisLNA to plasmid ratio of only 21:1. Although this first conceptual report does not address the utility of bisLNA for the targeting of DNA in a chromosomal context, it shows bisLNA as a promising candidate for interfering also with cellular genes.
Harmful algal blooms (HABs) are a serious public health risk in coastal waters. As the intensity and frequency of HABs continue to rise, new methods of detection are needed for reliable identification. Herein, we developed a high-throughput, multiplex, bead array technique for the detection of the dinoflagellates Karenia brevis and Karenia mikimotoi. The method combined the Luminex detection system with two novel technologies: locked nucleic acid–modified oligonucleotides (LNA) and Mirus Label IT® nucleic acid technology. To study the feasibility of the method, we evaluated the performance of modified and unmodified LNA probes with amplicon targets that were biotin labeled with two different strategies: direct chemical labeling (Mirus Label IT) versus enzymatic end-labeling (single biotinylated primer). The results illustrated that LNA probes hybridized to complementary single-stranded DNA with better affinity and displayed higher fluorescence intensities than unmodified oligonucleotide DNA probes. The latter effect was more pronounced when the assay was carried out at temperatures above 53°C degree. As opposed to the enzymatic 5′ terminal labeling technique, the chemical-labeling method enhanced the level of fluorescence by as much as ~83%. The detection limits of the assay, which were established with LNA probes and Mirus Label IT system, ranged from 0.05 to 46 copies of rRNA. This high-throughput method, which represents the first molecular detection strategy to integrate Luminex technology with LNA probes and Mirus Label IT, can be adapted for the detection of other HABs and is well suited for the monitoring of red tides at pre-blooming and blooming conditions.
Locked nucleic acids (LNA; symbols of bases, +A, +C,
+G, and +T) are introduced into chemically synthesized oligonucleotides
to increase duplex stability and specificity. To understand these
effects, we have determined thermodynamic parameters of consecutive
LNA nucleotides. We present guidelines for the design of LNA oligonucleotides
and introduce free online software that predicts the stability of
any LNA duplex oligomer. Thermodynamic analysis shows that the single
strand–duplex transition is characterized by a favorable enthalpic
change and by an unfavorable loss of entropy. A single LNA modification
confines the local conformation of nucleotides, causing a smaller,
less unfavorable entropic loss when the single strand is restricted
to the rigid duplex structure. Additional LNAs adjacent to the initial
modification appear to enhance stacking and H-bonding interactions
because they increase the enthalpic contributions to duplex stabilization.
New nearest-neighbor parameters correctly forecast the positive and
negative effects of LNAs on mismatch discrimination. Specificity is
enhanced in a majority of sequences and is dependent on mismatch type
and adjacent base pairs; the largest discriminatory boost occurs for
the central +C·C mismatch within the +T+C+C sequence and the
+A·G mismatch within the +T+A+G sequence. LNAs do not affect
specificity in some sequences and even impair it for many +G·T
and +C·A mismatches. The level of mismatch discrimination decreases
the most for the central +G·T mismatch within the +G+G+C sequence
and the +C·A mismatch within the +G+C+G sequence. We hypothesize
that these discrimination changes are not unique features of LNAs
but originate from the shift of the duplex conformation from B-form
Development of conformationally restricted nucleotide building blocks continues to attract considerable interest due to their successful use within antisense, antigene and other gene-targeting strategies. Locked nucleic acid (LNA) and its diastereomer α-L-LNA are two interesting examples hereof. Oligonucleotides modified with these units display greatly increased affinity toward nucleic acid targets, improved binding specificity and enhanced enzymatic stability relative to unmodified strands. Here, we present the synthesis and biophysical characterization of oligodeoxyribonucleotides (ONs) modified with 2′-amino-α-L-LNA adenine monomers W–Z. The synthesis of target phosphoramidites 1–4 initiates from pentafuranose 5, which upon Vorbrüggen glycosylation, O2′-deacylation, O2′-activation and C2′-azide introduction yields nucleoside 8. A one-pot tandem Staudinger/intramolecular nucleophilic substitution converts 8 into 2′-amino-α-L-LNA adenine intermediate 9, which after a series of non-trivial protecting group manipulations affords key intermediate 15. Subsequent chemoselective N2′-functionalization and O3′-phosphitylation gives targets 1–4 in ~1–3% overall yield over eleven steps from 5. ONs modified with pyrene-functionalized 2′-amino-α-L-LNA adenine monomers X-Z display greatly increased affinity toward DNA targets (ΔTm/modification up to +14 °C). Results from absorption and fluorescence spectroscopy suggest that the duplex stabilization is a result of pyrene intercalation. These characteristics render N2′-pyrene-functionalized 2′-amino-α-L-LNA of considerable interest for DNA-targeting applications.
To take full advantage of locked nucleic acid (LNA) based molecular beacons (LNA-MBs) for a variety of applications including analysis of complex samples and intracellular monitoring, we have systematically synthesized a series of DNA/LNA chimeric MBs and studied the effect of DNA/LNA ratio in MBs on their thermodynamics, hybridization kinetics, protein binding affinity and enzymatic resistance. It was found that the LNA bases in a MB stem sequence had a significant effect on the stability of the hair-pin structure. The hybridization rates of LNA-MBs were significantly improved by lowering the DNA/LNA ratio in the probe, and most significantly, by having a shared-stem design for the LNA-MB to prevent sticky-end pairing. It was found that only MB sequences with DNA/LNA alternating bases or all LNA bases were able to resist nonspecific protein binding and DNase I digestion. Additional results showed that a sequence consisting of a DNA stretch less than three bases between LNA bases was able to block RNase H function. This study suggested that a shared-stem MB with a 4 base-pair stem and alternating DNA/LNA bases is desirable for intracellular applications as it ensures reasonable hybridization rates, reduces protein binding and resists nuclease degradation for both target and probes. These findings have implications on the design of LNA molecular probes for intracellular monitoring application, disease diagnosis and basic biological studies.
Detection of unculturable bacteria and their localization in the host, by fluorescent in-situ hybridization (FISH), is a powerful technique in the study of host-bacteria interaction. FISH probes are designed to target the 16 s rRNA region of the bacteria to be detected. LNA probes have recently been used in FISH studies and proven to be more efficient. To date no report has employed LNA probes for FISH detection of bacterial endosymbiont in the whole mount tissues. Further, though speculated, bacteriocytes have not been reported from males of Bemisia tabaci.
In this study, we compared the efficiency in detecting bacteria by fluorescent DNA oligonucleotides versus modified probes containing Locked Nucleic Acid (LNA) substitution in their structure. We used the insect Bemisia tabaci as the experimental material since it carried simultaneous infection by two bacteria: one a primary endosymbiont, Portiera (and present in more numbers) while the other a secondary endosymbiont Arsenophonus (and present in less numbers). Thus a variation in the abundance of bacteria was expected. While detecting both the bacteria, we found a significant increase in the signal whenever LNA probes were used. However, the difference was more pronounced in detecting the secondary endosymbiont, wherein DNA probes gave weak signals when compared to LNA probes. Also, signal to noise ratio for LNA probes was higher than DNA probes. We found that LNA considerably improved sensitivity of FISH, as compared to the commonly used DNA oligonucleotide probe.
By employing LNA probes we could detect endosymbiotic bacteria in males, which have never been reported previously. We were able to detect bacteriocytes containing Portiera and Arsenophonus in the males of B. tabaci. Thus, employing LNA probes at optimized conditions will help to significantly improve detection of bacteria at the lowest concentration and may give a comprehensible depiction about their specific distribution within samples.
The available reagents for the attachment of functional moieties to plasmid DNA are limiting. Most reagents bind plasmid DNA in a non-sequence- specific manner, with undefined stoichiometry, and affect DNA charge and delivery properties or involve chemical modifications that abolish gene expression. The design and ability of oligonucleotides (ODNs) containing locked nucleic acids (LNAs) to bind supercoiled, double-stranded plasmid DNA in a sequence-specific manner are described for the first time. The main mechanism for LNA ODNs binding plasmid DNA is demonstrated to be by strand displacement. LNA ODNs are more stably bound to plasmid DNA than similar peptide nucleic acid (PNA) ‘clamps’ for procedures such as particle-mediated DNA delivery (gene gun). It is shown that LNA ODNs remain associated with plasmid DNA after cationic lipid-mediated transfection into mammalian cells. LNA ODNs can bind to DNA in a sequence-specific manner so that binding does not interfere with plasmid conformation or gene expression. Attachment of CpG-based immune adjuvants to plasmid by ‘hybrid’ phosphorothioate–LNA ODNs induces tumour necrosis factor-α production in the macrophage cell line RAW264.7. This observation exemplifies an important new, controllable methodology for adding functionality to plasmids for gene delivery and DNA vaccination.
The simultaneous extraction of plant organelle (mitochondria and plastid) genes during the DNA extraction step is a major limitation in investigating the community structures of bacteria associated with plants because organelle SSU rRNA genes are easily amplified by PCR using primer sets that are specific to bacteria. To inhibit the amplification of organelle genes, the locked nucleic acid (LNA) oligonucleotide–PCR clamping technique was applied to selectively amplify bacterial SSU rRNA genes by PCR. LNA oligonucleotides, the sequences of which were complementary to mitochondria and plastid genes, were designed by overlapping a few bases with the annealing position of the bacterial primer and converting DNA bases into LNA bases specific to mitochondria and plastids at the shifted region from the 3′ end of the primer-binding position. PCR with LNA oligonucleotides selectively amplified the bacterial genes while inhibited that of organelle genes. Denaturing gradient gel electrophoresis (DGGE) analysis revealed that conventional amplification without LNA oligonucleotides predominantly generated DGGE bands from mitochondria and plastid genes with few bacterial bands. In contrast, additional bacterial bands were detected in DGGE patterns, the amplicons of which were prepared using LNA oligonucleotides. These results indicated that the detection of bacterial genes had been screened by the excessive amplification of the organelle genes. Sequencing of the bands newly detected by using LNA oligonucleotides revealed that their similarity to the known isolated bacteria was low, suggesting the potential to detect novel bacteria. Thus, application of the LNA oligonucleotide–PCR clamping technique was considered effective for the selective amplification of bacterial genes from extracted DNA containing plant organelle genes.
plant organelle; bacteria; community structure; SSU rRNA gene; locked nucleic acid (LNA) oligonucleotide–PCR clamping
Locked nucleic acids (LNAs) and double-stranded small interfering RNAs (siRNAs) are rather new promising antisense molecules for cell culture and in vivo applications. Here, we compare LNA–DNA–LNA gapmer oligonucleotides and siRNAs with a phosphorothioate and a chimeric 2′-O-methyl RNA–DNA gapmer with respect to their capacities to knock down the expression of the vanilloid receptor subtype 1 (VR1). LNA–DNA–LNA gapmers with four or five LNAs on either side and a central stretch of 10 or 8 DNA monomers in the center were found to be active gapmers that inhibit gene expression. A comparative co-transfection study showed that siRNA is the most potent inhibitor of VR1–green fluorescent protein (GFP) expression. A specific inhibition was observed with an estimated IC50 of 0.06 nM. An LNA gapmer was found to be the most efficient single-stranded antisense oligonucleotide, with an IC50 of 0.4 nM being 175-fold lower than that of commonly used phosphorothioates (IC50 ∼70 nM). In contrast, the efficiency of a 2′-O-methyl-modified oligonucleotide (IC50 ∼220 nM) was 3-fold lower compared with the phosphorothioate. The high potency of siRNAs and chimeric LNA–DNA oligonucleotides make them valuable candidates for cell culture and in vivo applications targeting the VR1 mRNA.
Antisense-mediated modulation of pre-mRNA splicing is an attractive therapeutic strategy for genetic diseases. Currently, there are few examples of modulation of pre-mRNA splicing using locked nucleic acid (LNA) antisense oligonucleotides, and, in particular, no systematic study has addressed the optimal design of LNA-based splice-switching oligonucleotides (LNA SSOs). Here, we designed a series of LNA SSOs complementary to the human dystrophin exon 58 sequence and evaluated their ability to induce exon skipping in vitro using reverse transcription-polymerase chain reaction. We demonstrated that the number of LNAs in the SSO sequence and the melting temperature of the SSOs play important roles in inducing exon skipping and seem to be key factors for designing efficient LNA SSOs. LNA SSO length was an important determinant of activity: a 13-mer with six LNA modifications had the highest efficacy, and a 7-mer was the minimal length required to induce exon skipping. Evaluation of exon skipping activity using mismatched LNA/DNA mixmers revealed that 9-mer LNA SSO allowed a better mismatch discrimination. LNA SSOs also induced exon skipping of endogenous human dystrophin in primary human skeletal muscle cells. Taken together, our findings indicate that LNA SSOs are powerful tools for modulating pre-mRNA splicing.
We have screened 47 locked nucleic acid (LNA) antisense oligonucleotides (ASOs) targeting conserved (>95% homology) sequences in the hepatitis C virus (HCV) genome using the subgenomic HCV replicon assay and generated both antiviral (50% effective concentration [EC50]) and cytotoxic (50% cytotoxic concentration [CC50]) dose-response curves to allow measurement of the selectivity index (SI). This comprehensive approach has identified an LNA ASO with potent antiviral activity (EC50 = 4 nM) and low cytotoxicity (CC50 >880 nM) targeting the 25- to 40-nucleotide region (nt) of the HCV internal ribosome entry site (IRES) containing the distal and proximal miR-122 binding sites. LNA ASOs targeting previously known accessible regions of the IRES, namely, loop III and the initiation codon in loop IV, had poor SI values. We optimized the LNA ASO sequence by performing a 1-nucleotide walk through the 25- to 40-nt region and show that the boundaries for antiviral efficacy are extremely precise. Furthermore, we have optimized the format for the LNA ASO using different gapmer and mixomer patterns and show that RNase H is required for antiviral activity. We demonstrate that RNase H-refractory ASOs targeting the 25- to 40-nt region have no antiviral effect, revealing important regulatory features of the 25- to 40-nt region and suggesting that RNase H-refractory LNA ASOs can act as potential surrogates for proviral functions of miR-122. We confirm the antisense mechanism of action using mismatched LNA ASOs. Finally, we have performed pharmacokinetic experiments to demonstrate that the LNA ASOs have a very long half-life (>5 days) and attain hepatic maximum concentrations >100 times the concentration required for in vitro antiviral activity.
The influence of locked nucleic acid (LNA) residues on the thermodynamic properties of 2′-O-methyl RNA/RNA heteroduplexes is reported. Optical melting studies indicate that LNA incorporated into an otherwise 2′-O-methyl RNA oligonucleotide usually, but not always, enhances the stabilities of complementary duplexes formed with RNA. Several trends are apparent, including: (i) a 3′ terminal U LNA and 5′ terminal LNAs are less stabilizing than interior and other 3′ terminal LNAs; (ii) most of the stability enhancement is achieved when LNA nucleotides are separated by at least one 2′-O-methyl nucleotide; and (iii) the effects of LNA substitutions are approximately additive when the LNA nucleotides are separated by at least one 2′-O-methyl nucleotide. An equation is proposed to approximate the stabilities of complementary duplexes formed with RNA when at least one 2′-O-methyl nucleotide separates LNA nucleotides. The sequence dependence of 2′-O-methyl RNA/RNA duplexes appears to be similar to that of RNA/RNA duplexes, and preliminary nearest-neighbor free energy increments at 37°C are presented for 2′-O-methyl RNA/RNA duplexes. Internal mismatches with LNA nucleotides significantly destabilize duplexes with RNA.
The design of antisense oligonucleotides containing locked nucleic acids (LNA) was optimized and compared to intensively studied DNA oligonucleotides, phosphorothioates and 2′-O-methyl gapmers. In contradiction to the literature, a stretch of seven or eight DNA monomers in the center of a chimeric DNA/LNA oligonucleotide is necessary for full activation of RNase H to cleave the target RNA. For 2′-O-methyl gapmers a stretch of six DNA monomers is sufficient to recruit RNase H. Compared to the 18mer DNA the oligonucleotides containing LNA have an increased melting temperature of 1.5–4°C per LNA depending on the positions of the modified residues. 2′-O-methyl nucleotides increase the Tm by only <1°C per modification and the Tm of the phosphorothioate is reduced. The efficiency of an oligonucleotide in supporting RNase H cleavage correlates with its affinity for the target RNA, i.e. LNA > 2′-O-methyl > DNA > phosphorothioate. Three LNAs at each end of the oligonucleotide are sufficient to stabilize the oligonucleotide in human serum 10-fold compared to an unmodified oligodeoxynucleotide (from t1/2 = ∼1.5 h to t1/2 = ~15 h). These chimeric LNA/DNA oligonucleotides are more stable than isosequential phosphorothioates and 2′-O-methyl gapmers, which have half-lives of 10 and 12 h, respectively.
Genomes are becoming heavily annotated with important features. Analysis of these features often employs oligonucleotides that hybridize at defined locations. When the defined location lies in a poor sequence context, traditional design strategies may fail. Locked Nucleic Acid (LNA) can enhance oligonucleotide affinity and specificity. Though LNA has been used in many applications, formal design rules are still being defined. To further this effort we have investigated the effect of LNA on the performance of sequencing and PCR primers in AT-rich regions, where short primers yield poor sequencing reads or PCR yields. LNA was used in three positional patterns: near the 5′ end (LNA-5′), near the 3′ end (LNA-3′) and distributed throughout (LNA-Even). Quantitative measures of sequencing read length (Phred Q30 count) and real-time PCR signal (cycle threshold, CT) were characterized using two-way ANOVA. LNA-5′ increased the average Phred Q30 score by 60% and it was never observed to decrease performance. LNA-5′ generated cycle thresholds in quantitative PCR that were comparable to high-yielding conventional primers. In contrast, LNA-3′ and LNA-Even did not improve read lengths or CT. ANOVA demonstrated the statistical significance of these results and identified significant interaction between the positional design rule and primer sequence.
Locked nucleic acids (LNAs) are synthetic nucleic acid analogs that bind to complementary target molecules (DNA, RNA or LNA) with very high affinity. At the same time, this binding affinity is decreased substantially when the hybrids thus formed contain even a single mismatched base pair. We have exploited these properties of LNA probes to develop a new method for single nucleotide polymorphism genotyping. In this method, very short (hexamer or heptamer) LNA probes are labeled with either rhodamine or hexachlorofluorescein (HEX), and their hybridization to target DNAs is followed by measuring the fluorescence polarization (FP) of the dyes. The formation of perfectly complementary double-stranded hybrids gives rise to significant FP increases, whereas the presence of single mismatches results in very small or no changes of this parameter. Multiplexing of the assay can be achieved by using differentially labeled wild-type and mutant specific probes in the same solution. The method is homogeneous, and because of the use of extremely short LNA probes, the generation of a universal set of genotyping reagents is possible.