---
title: "Know your oligo mod: Locked nucleic acids"
description: Learn how locked nucleic acids work and how they are being used in qPCR probes and primers and in therapeutic development in this free blog.
image: https://blog.biosearchtech.com/hubfs/Scientist%20reading%20locked%20nucleic%20acid%20blog.png
---
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[Blog](https://blog.biosearchtech.com) / Know your oligo mod: Locked nucleic acids
# Know your oligo mod: Locked nucleic acids
[Oligo synthesis and modifications](https://blog.biosearchtech.com/topic/oligo-synthesis-and-modifications)
**Time to read:** 5 minute read
Originally published : Tue, October 29, 2024 @ 6:40 PM
Locked nucleic acids are an increasingly popular modification for tailoring and improving the performance of oligonucleotides in a variety of ways. As part of our Know Your Oligo Mod series, we look at how these modified bases work and how they are being used in qPCR probes and primers and in therapeutic development.
#### An introduction to locked nucleic acids
Locked nucleic acids are modified nucleotides with a methylene group that bridges the 2ʹ-O and 4ʹ-C of the ribose ring.1 The bridging bond locks the flexible ribose ring in the conformation usually found in duplexes, reducing the entropy loss upon hybridisation. By making the thermodynamics more favourable, LNAs increase the Tm for oligonucleotide hybridisation.2
Like other 2ʹ modifications, LNAs increase stability against nucleases. Kurreck *et al.* found that three LNAs at both 3ʹ and 5ʹ ends offered a 10-fold increase in serum half-life compared to unmodified oligos.3
Another important advantage of LNAs is their ability to improve the discrimination of subtle mismatches, such as single nucleotide polymorphisms and transcript variants.4-6 While standard oligonucleotides can struggle to distinguish point mutations, a single base mismatch has a greater destabilising effect when a shorter LNA probe is forming a duplex.
#### Putting locked nucleic acids to use
Each LNA substitution in an oligo can increase the Tm of a duplex by up to 8 °C.7 This provides developers with a valuable degree of control to fine-tune the desired Tm and levels of specificity and sensitivity.
While this can involve more iterations of testing to optimise than an [MGB Probe](https://www.biosearchtech.com/products/pcr-reagents-kits-and-instruments/pcr-probes-and-assays/custom-dna-probes-for-qpcr/minor-groove-binder-mgb-probes), [LNA Probes](https://www.biosearchtech.com/products/pcr-reagents-kits-and-instruments/pcr-probes-and-assays/custom-dna-probes-for-qpcr/locked-nucleic-acid-probes) can offer improved target discrimination. The greater stability and stronger binding of LNA oligos allow developers to create shorter-length probes, which are more specific and have better quenching.8
The advantages of LNA oligos make them ideal for applications that require high specificity:
- Template switching oligos for constructing cDNA libraries in RNA sequencing studies9
- Multiplex qPCR assays, avoiding interference across reactions
- Targeting AT-rich sequences
- Challenging samples, such as formalin-fixed paraffin-embedded (FFPE) tissue10
#### Therapeutic applications of LNA oligos
Outside sequencing and molecular diagnostics, LNAs are also being applied in therapeutic approaches, such as [antisense oligonucleotides (ASOs)](https://blog.biosearchtech.com/whats-the-latest-in-aso-and-sirna-technologies). These molecules combine a mixture of RNA and DNA nucleosides, which when binding to the complementary RNA, recruit RNase H to degrade the target.
LNA oligos are attracting interest in targeting microRNAs, small but important regulatory non-coding RNAs. The short sequences and similarity between miRNAs make them challenging targets, which LNAs can help to overcome when designing inhibitors.
Candidate therapeutics have been developed to treat myocardial infarction, pulmonary fibrosis, lymphoma and hepatitis C. However, none of these have been able to successfully reach late-stage clinical trials, as of 2024. Some of the compounds tested include:
- MGN-1374, an LNA ASO designed to target the miR-15 family in the preclinical phase for **postmyocardial infarction** treatment.11
- MRG-110, an LNA ASO against miR-92a-3p for the treatment of **heart failure** and to improve wound healing. It was tested in healthy volunteers in phase I trials, but has stopped clinical development.12,13
- Cobomarsen (MRG-106), an LNA-based oligo that aimed to inhibit the activity of miR-155 in several **lymphoma** subtypes. While the phase I trial was completed, two of the phase II studies were terminated and development was discontinued.13,14
- Miravirsen, a 15-mer LNA-modified ASO which targeted miR-122 as a treatment for **Hepatitis C** infection. This was discontinued after the development of effective antiviral treatments.
- LNA-i-miR-221, a 13-mer LNA inhibitor of miR-221 was tested in a phase I clinical trial in **cancer**.15
- LNA-anti-miR-21, an inhibitor of miR-21 designed to treat **melanoma** in preclinical development.16
- MB_1114, an LNA ASO in preclinical development for inhibiting mRNA coding for the protein mLDHB to treat **autoimmune myocarditis**.17
- LNA-anti-miR-23b, an inhibitor of miR-23b to treat **liver cancer**, is currently in preclinical development.18
For therapeutics, one of the key challenges with LNA oligos is liver toxicity. However, various strategies have been explored to mitigate this side effect, including altering the number of LNA bases, changing the bases and incorporating nucleobase derivatives, such as 5-hydroxycytosine, 2-thiothymine and 8-bromoguanine.19,20 Also, using different bridging groups, such as constrained methyl- and ethyl-bridges as well as guanidine-bridges, have shown promise in reducing hepatotoxicity.21,22 These offer potential avenues for developing safer LNA-based therapeutics.
In conclusion, locked nucleic acids present many opportunities for developing high-affinity oligonucleotides, as probes, miRNA inhibitors and much more.
#### Order custom oligos with locked nucleic acids
LGC Biosearch Technologies has a range of products available to meet your locked nucleic acids needs:
- Dual-HPLC purified, MS and uHPLC verified LNA Probes with access to a wide range of fluorophores for multiplexing and the flexibility to add up to seven LNA bases in the probe
- Custom oligos and primers with up to 20 internal LNA modifications
- A range of [LNA phosphoramidites and CPG](https://shop.biosearchtech.com/nucleic-acid-chemistry-reagents-and-instruments/modified-oligo-synthesis-reagents/backbone-modifications/locked-nucleic-acids-%28lna%29/c/locked-nucleic-acids-%28lna%29) for in-house synthesis.
[](https://cta-redirect.hubspot.com/cta/redirect/54061/bdb08320-5b5f-440f-a59b-328046851e17)
---
#### Know your oligo mod series
- [5-Nitroindole – a universal base oligo modification](https://blog.biosearchtech.com/5-nitroindole-universal-base-oligo-modification)
- [BHQ® (Black Hole Quencher®) non-fluorescent quenchers](https://blog.biosearchtech.com/know-your-oligo-mod-bhq-black-hole-quencher-dyes)
- [Thio C6 linker](https://blog.biosearchtech.com/know-your-oligo-mod-thio-c6-linker)
- [3' Spacer C3](https://blog.biosearchtech.com/know-your-oligo-mod-spacer-c3)
- [Phosphorothioate bonds](https://blog.biosearchtech.com/know-your-oligo-mod-phosphorothioate-bonds)
- [2'-O-methoxyethyl (2'-MOE)](https://blog.biosearchtech.com/know-your-oligo-mod-2%CA%B9-moe)
---
References
1. Obika S *et al.* Synthesis of 2′-O,4′-C-methyleneuridine and -cytidine. Novel bicyclic nucleosides having a fixed C3, -endo sugar puckering. *Tetrahedron Letters*. 1997; 38 (50): 8735–8738. doi:[10.1016/S0040-4039(97)10322-7](https://doi.org/10.1016%2FS0040-4039%2897%2910322-7)
2. You Y, Moreira BG, Behlke MA, *et al*. Design of LNA probes that improve mismatch discrimination. *Nucleic Acids Res*. 2006;34(8):e60. doi: [10.1093/nar/gkl175](https://doi.org/10.1093/nar/gkl175)
3. Kurreck J *et al.* Design of antisense oligonucleotides stabilized by locked nucleic acids. *Nucleic Acids Res. *2002;30:1911–1918. doi: [10.1093/nar/30.9.1911](https://doi.org/10.1093/nar/30.9.1911)
4. Mouritzen P, Nielsen AT, Pfundheller HM *et al*. Single nucleotide polymorphism genotyping using locked nucleic acid (LNA). *Exper Rev Mol Diagn*. 2003;3(1):27-38. doi: [10.1586/14737159.3.1.27](https://doi.org/10.1586/14737159.3.1.27)
5. Latorra D, Campbell K, Wolter A, Hurley JM. Enhanced allele-specific PCR discrimination in SNP genotyping using 3ʹ locked nucleic acid (LNA) primers.* Hum. Mutat.* 2003; 22(1):79-85. doi: [10.1002/humu.10228](https://doi.org/10.1002/humu.10228)
6. Simeonov A. Single nucleotide polymorphism genotyping using short, fluorescently labeled locked nucleic acid (LNA) probes and fluorescence polarization detection. *Nucleic Acids Res*. 2002; 30(17):91e-91. doi: [10.1093/nar/gnf090](https://doi.org/10.1093/nar/gnf090)
7. Christensen *et al.* Stopped-flow kinetics of locked nucleic acid (LNA)-oligonucleotide duplex formation: studies of LNA-DNA and DNA-DNA interactions. *Biochemical J.* 2001; 354(3):481-484. doi: [10.1042/bj3540481](https://doi.org/10.1042/bj3540481)
8. Ugozzoli LA, Latorra D, Puckett R, *et al*. Real-time genotyping with oligonucleotide probes containing locked nucleic acids. *Anal Biochem*. 2004;324(1):143-152. doi: [10.1016/j.ab.2003.09.003](https://doi.org/10.1016/j.ab.2003.09.003)
9. Harbers M, Kato S, de Hoon M, *et al*. Comparison of RNA- or LNA-hybrid oligonucleotides in template-switching reactions for high-speed sequencing library preparation. *BMC Genomics*. 2013;14:665. doi: [10.1186/1471-2164-14-665](https://doi.org/10.1186/1471-2164-14-665)
10. Paulsen IW *et al*. A novel approach for microRNA in situ hybridization using locked nucleic acid probes. *Scientific Reports*. 2021; 11:4504 doi:[10.1038/s41598-021-83888-5](https://doi.org/10.1038/s41598-021-83888-5)
11. Iacomino G. miRNAs: The Road from Bench to Bedside. [*Genes (Basel)*.](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9957002/) 2023; 14(2): 314. doi: [10.3390/genes14020314](https://doi.org/10.3390%2Fgenes14020314)
12. Abplanalp WT *et al*. Efficiency and Target Derepression of Anti-miR-92a: Results of a First in Human Study. *Nucleic acid therapeutics*. 2020 doi:[10.1089/nat.2020.0871](https://www.liebertpub.com/doi/10.1089/nat.2020.0871)
13. Khorkova O, Stahl J, Joji A *et al.* Amplifying gene expression with RNA-targeted therapeutics. *Nat Rev Drug Discov.* 2023; 22, 539–561. doi: [10.1038/s41573-023-00704-7](https://doi.org/10.1038/s41573-023-00704-7)
14. Querfeld C *et al.* Preliminary Results of a Phase 1 Trial Evaluating MRG-106, a Synthetic microRNA Antagonist (LNA antimiR) of microRNA-155, in Patients with CTCL. *Blood* 2016; 128, 1829 doi:[ 10.1182/blood.V128.22.1829.1829](https://doi.org/10.1182/blood.V128.22.1829.1829)
15. Tassone P *et al.* Safety and activity of the first-in-class locked nucleic acid (LNA) miR-221 selective inhibitor in refractory advanced cancer patients: a first-in-human, phase 1, open-label, dose-escalation study. *J Hematol Oncol* 2023; 16, 68. doi: [10.1186/s13045-023-01468-8](https://doi.org/10.1186/s13045-023-01468-8)
16. Javanmard SH *et al.* Therapeutic inhibition of microRNA-21 (miR-21) using locked-nucleic acid (LNA)-anti-miR and its effects on the biological behaviors of melanoma cancer cells in preclinical studies. *Cancer Cell Int* 2020; 20, 384. doi: [10.1186/s12935-020-01394-6](https://doi.org/10.1186/s12935-020-01394-6)
17. Bockstahler M *et al.* LNA oligonucleotide mediates an anti-inflammatory effect in autoimmune myocarditis via targeting lactate dehydrogenase B. *Immunology* 2022; 165(2):158-170 doi: [10.1111/imm.13421](https://doi.org/10.1111/imm.13421)
18. Najafi Z *et al.* LNA Inhibitor in microRNA miR-23b as a Potential Anti-proliferative Option in Human Hepatocellular Carcinoma. *Journal of Gastrointestinal Cancer* 2020; 51:109-115 doi:[10.1007/s12029-019-00215-y](https://link.springer.com/article/10.1007/s12029-019-00215-y)
19. Hagedorn PH *et al.* Locked nucleic acid: modality, diversity, and drug discovery. Drug Discovery Today 2018; 23(1):101-114 doi:[10.1016/j.drudis.2017.09.018](https://doi.org/10.1016/j.drudis.2017.09.018)
20. Yoshida T, Morihiro K, Naito Y, *et al.* Identification of nucleobase chemical modifications that reduce the hepatotoxicity of gapmer antisense oligonucleotides. *Nucleic Acids Res.* 2022;50(13):7224-7234. doi:[10.1093/nar/gkac562](https://doi.org/10.1093/nar/gkac562)
21. Xiong H, Veedu RN, Diermeier SD Recent Advances in Oligonucleotide Therapeutics in Oncology. *International Journal of Molecular Sciences*, 2021; *22*(7). doi:[10.3390/ijms22073295](https://doi.org/10.3390/ijms22073295)
22. Sasaki T, Hirakawa Y, Yamairi F *et al.* Altered Biodistribution and Hepatic Safety Profile of a Gapmer Antisense Oligonucleotide Bearing Guanidine-Bridged Nucleic Acids. *Nucleic Acid Ther.* 2022;32(3):177-184. doi:[10.1089/nat.2021.003](https://doi.org/10.1089/nat.2021.0034)
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