Locked nucleic acid

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Chemical structure of an LNA monomer an additional bridge bonds the 2' oxygen and the 4' carbon of the pentose LNASchem.svg
Chemical structure of an LNA monomer an additional bridge bonds the 2' oxygen and the 4' carbon of the pentose

A locked nucleic acid (LNA), also known as bridged nucleic acid (BNA), [1] and often referred to as inaccessible RNA, is a modified RNA nucleotide in which the ribose moiety is modified with an extra bridge connecting the 2' oxygen and 4' carbon. The bridge "locks" the ribose in the 3'-endo (North) conformation, which is often found in the A-form duplexes. This structure provides for increased stability against enzymatic degradation. [2] [3] [4] [5] LNA also offers improved specificity and affinity in base-pairing as a monomer or a constituent of an oligonucleotide. [6] LNA nucleotides can be mixed with DNA or RNA residues in a oligonucleotide.

Contents

Synthesis

Obika et al. were the first to chemically synthesize LNA in 1997, [7] independently followed by Jesper Wengel's group in 1998. [8] This became possible after Zamecnick and Stephenson laid the groundwork on the possibility of oligonucleotides being great agents for controlling gene expression in 1978. [9] To date, two different approaches, referred to as linear and convergent strategies respectively, have been shown to produce high yield and efficient LNAs. The linear strategy of synthesis was first detailed in the works of Obika et al. [7] In this approach, uridine (or any readily available RNA nucleoside) can be used as the starting material. The convergent strategy requires the synthesis of a sugar intermediate which serves a glycosyl donor necessary for coupling with nucleobases. Commonly, D-glucose is used to produce the sugar intermediate which is subsequently reacted with nucleobases using a modified Vorbrügen procedure allowing for stereoselective coupling. [10]

The addition of different moieties has remained a possibility with the maintenance of key physicochemical properties like the high affinity and specificity evident in the originally synthesized LNA. [8] Such oligomers are synthesized chemically and are commercially available.

Incorporation into DNA/RNA

LNA can be incorporated into DNA and RNA using the promiscuity of certain DNA and RNA polymerases. Phusion DNA polymerase, a commercially designed enzyme based on a Pfu DNA polymerase, efficiently incorporates LNA into DNA. [11]

Properties

LNA offers enhanced biostability compared to biological nucleic acids. LNA modified oligonucleotides have demonstrated improved thermodynamics in hybridization to RNA, ssDNA, and dsDNA. [11]

Applications

LNAzymes

DNAzymes can be modified to include LNA residues, producing LNAzymes (LNA-modified DNAzymes). These modified oligonucleotides, like their DNAzyme relatives, are generally endonucleases that bind to specific RNA target sequences and cleave the phosphodiester bond that exists between the nucleotides. [12] However, they demonstrate more efficient cleavage of phosphodiester bonds compared to their unmodified counterparts. [13] Modification of the substrate recognition arms of DNAzymes with LNA monomers yields a LNAzyme which recognizes coxsackievirus A21 (CAV-21) and cleaves its RNA target sequence similar to one in the 5' untranslated region (5' UTR) of the human rhinovirus-14 (HRV-14); a sequence unrecognized by unmodified DNAzymes. [14]

Therapeutics

Using LNA based oligonucleotides therapeutically is an emerging field in biotechnology. [15] A variety of LNA oligonucleotides have been assessed for their pharmacokinetic and toxicity profiles. Studies concluded that LNA toxicity is generally independent of oligonucleotide sequence, and displays a preferential safety profile for translatable therapeutic applications. [8]

LNA has been investigated for its therapeutic properties in treating cancers and infectious diseases. A locked nucleic acid phosphorothioate antisense molecule, termed SPC2996, has been developed to target the mRNA coding for Bcl-2 oncoprotein, a protein that inhibits apoptosis in chronic lymphocytic leukemia cells (CLL). Phase I and II clinical trials demonstrated a dose dependent reduction in circulating CLL cells in approximately 30% of the sample population, suggesting further investigation into SPC2996. [16]

LNA has also been applied to Miravirsen, an experimental therapeutic intended for the treatment of Hepatitis C, constituting a 15-nucleotide phosphorothioate sequence with binding specificity for MiR-122 (a miRNA expressed in hepatocytes). [17] [18]

Detection and diagnosis

Allele-specific PCR using LNA allows for the design of shorter primers, without compromising binding specificity. [19]

LNA has been incorporated in fluorescence in situ hybridization (FISH). [20] FISH is a common technique used to visualize genetic material in a variety of cells, but studies noted that this technique has been limited by low probe hybridization efficiency. Conversely, LNA-incorporated probes demonstrated increased hybridization efficiency in both DNA and RNA. The improved efficiency of LNA-incorporated FISH has resulted in FISH analysis of the human chromosome, several types of non-human cells, and microarrays. [20]

LNA genotyping assays have been conducted as well, specifically to detect a mutation in apolipoprotein B. [20]

For its high affinity for mismatch discrimination, LNA has been studied for its applications in diagnostic tools. Immobilized LNA probes have been introduced in a multiplex SNP genotyping assay. [15]

Gene editing

LNA-modified ssODNs (synthetic single-stranded DNA oligonucleotides) can be used like ordinary ssODNs for single-base gene editing. Using LNA at or close to the intended site of modification offers evasion of DNA mismatch repair due to the higher thermodynamic stability it has. [21]

Related Research Articles

<span class="mw-page-title-main">Nucleic acid</span> Class of large biomolecules essential to all known life

Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main classes of nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). If the sugar is ribose, the polymer is RNA; if the sugar is the ribose derivative deoxyribose, the polymer is DNA.

Nucleoside Any of several glycosylamines comprising a nucleobase and a sugar molecule

Nucleosides are glycosylamines that can be thought of as nucleotides without a phosphate group. A nucleoside consists simply of a nucleobase and a five-carbon sugar whereas a nucleotide is composed of a nucleobase, a five-carbon sugar, and one or more phosphate groups. In a nucleoside, the anomeric carbon is linked through a glycosidic bond to the N9 of a purine or the N1 of a pyrimidine. Nucleotides are the molecular building-blocks of DNA and RNA.

Peptide nucleic acid Biological molecule

Peptide nucleic acid (PNA) is an artificially synthesized polymer similar to DNA or RNA.

Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, research, and forensics. Commonly made in the laboratory by solid-phase chemical synthesis, these small bits of nucleic acids can be manufactured as single-stranded molecules with any user-specified sequence, and so are vital for artificial gene synthesis, polymerase chain reaction (PCR), DNA sequencing, molecular cloning and as molecular probes. In nature, oligonucleotides are usually found as small RNA molecules that function in the regulation of gene expression, or are degradation intermediates derived from the breakdown of larger nucleic acid molecules.

Antisense therapy is a form of treatment that uses antisense oligonucleotides (ASOs) to target messenger RNA (mRNA). ASOs are capable of altering mRNA expression through a variety of mechanisms, including ribonuclease H mediated decay of the pre-mRNA, direct steric blockage, and exon content modulation through splicing site binding on pre-mRNA. Several ASOs have been approved in the United States, the European Union, and elsewhere.

In molecular biology, a hybridization probe(HP) is a fragment of DNA or RNA of usually 15–10000 nucleotide long which can be radioactively or fluorescently labeled. HP can be used to detect the presence of nucleotide sequences in analyzed RNA or DNA that are complementary to the sequence in the probe. The labeled probe is first denatured into single stranded DNA (ssDNA) and then hybridized to the target ssDNA or RNA immobilized on a membrane or in situ.

Deoxyribozymes, also called DNA enzymes, DNAzymes, or catalytic DNA, are DNA oligonucleotides that are capable of performing a specific chemical reaction, often but not always catalytic. This is similar to the action of other biological enzymes, such as proteins or ribozymes . However, in contrast to the abundance of protein enzymes in biological systems and the discovery of biological ribozymes in the 1980s, there is only little evidence for naturally occurring deoxyribozymes. Deoxyribozymes should not be confused with DNA aptamers which are oligonucleotides that selectively bind a target ligand, but do not catalyze a subsequent chemical reaction.

Threose nucleic acid (TNA) is an artificial genetic polymer in which the natural five-carbon ribose sugar found in RNA has been replaced by an unnatural four-carbon threose sugar. Invented by Albert Eschenmoser as part of his quest to explore the chemical etiology of RNA, TNA has become an important synthetic genetic polymer (XNA) due to its ability to efficiently base pair with complementary sequences of DNA and RNA. However, unlike DNA and RNA, TNA is completely refractory to nuclease digestion, making it a promising nucleic acid analog for therapeutic and diagnostic applications.

Molecular beacon

Molecular beacons, or molecular beacon probes, are oligonucleotide hybridization probes that can report the presence of specific nucleic acids in homogenous solutions. Molecular beacons are hairpin-shaped molecules with an internally quenched fluorophore whose fluorescence is restored when they bind to a target nucleic acid sequence. This is a novel non-radioactive method for detecting specific sequences of nucleic acids. They are useful in situations where it is either not possible or desirable to isolate the probe-target hybrids from an excess of the hybridization probes.

In molecular biology and genetics, the sense of a nucleic acid molecule, particularly of a strand of DNA or RNA, refers to the nature of the roles of the strand and its complement in specifying a sequence of amino acids. Depending on the context, sense may have slightly different meanings. For example, negative-sense strand of DNA is equivalent to the template strand, whereas the positive-sense strand is the non-template strand whose nucleotide sequence is equivalent to the sequence of the mRNA transcript.

Systematic evolution of ligands by exponential enrichment Technique for producing oligonucleotides that specifically bind to a target

Systematic evolution of ligands by exponential enrichment (SELEX), also referred to as in vitro selection or in vitro evolution, is a combinatorial chemistry technique in molecular biology for producing oligonucleotides of either single-stranded DNA or RNA that specifically bind to a target ligand or ligands. These single-stranded DNA or RNA are commonly referred to as aptamers. Although SELEX has emerged as the most commonly used name for the procedure, some researchers have referred to it as SAAB and CASTing SELEX was first introduced in 1990. In 2015, a special issue was published in the Journal of Molecular Evolution in the honor of quarter century of the discovery of SELEX.

Oligonucleotide synthesis is the chemical synthesis of relatively short fragments of nucleic acids with defined chemical structure (sequence). The technique is extremely useful in current laboratory practice because it provides a rapid and inexpensive access to custom-made oligonucleotides of the desired sequence. Whereas enzymes synthesize DNA and RNA only in a 5' to 3' direction, chemical oligonucleotide synthesis does not have this limitation, although it is most often carried out in the opposite, 3' to 5' direction. Currently, the process is implemented as solid-phase synthesis using phosphoramidite method and phosphoramidite building blocks derived from protected 2'-deoxynucleosides, ribonucleosides, or chemically modified nucleosides, e.g. LNA or BNA.

Nucleic acid analogue Compound analogous to naturally occurring RNA and DNA

Nucleic acid analogues are compounds which are analogous to naturally occurring RNA and DNA, used in medicine and in molecular biology research. Nucleic acids are chains of nucleotides, which are composed of three parts: a phosphate backbone, a pentose sugar, either ribose or deoxyribose, and one of four nucleobases. An analogue may have any of these altered. Typically the analogue nucleobases confer, among other things, different base pairing and base stacking properties. Examples include universal bases, which can pair with all four canonical bases, and phosphate-sugar backbone analogues such as PNA, which affect the properties of the chain . Nucleic acid analogues are also called Xeno Nucleic Acid and represent one of the main pillars of xenobiology, the design of new-to-nature forms of life based on alternative biochemistries.

Santaris Pharma A/S was a biopharmaceutical company founded in 2003 in Copenhagen, Denmark. The company also had a branch in San Diego, California that opened in 2009. Created by a merger between Cureon and Pantheco, Santaris developed RNA-targeted medicines using a Locked Nucleic Acid (LNA) Drug Platform and Drug Development Engine.

Twisted intercalating nucleic acid (TINA) is a nucleic acid molecule that, when added to triplex-forming oligonucleotides (TFOs), stabilizes Hoogsteen triplex DNA formation from double-stranded DNA (dsDNA) and TFOs. Its ability to twist around a triple bond increases ease of intercalation within double stranded DNA in order to form triplex DNA. Certain configurations have been shown to stabilize Watson-Crick antiparallel duplex DNA. TINA-DNA primers have been shown to increase the specificity of binding in PCR. The use of TINA insertions in G-quadruplexes has also been shown to enhance anti-HIV-1 activity. TINA stabilized PT demonstrates improved sensitivity and specificity of DNA based clinical diagnostic assays.

A bridged nucleic acid (BNA) is a modified RNA nucleotide. They are sometimes also referred to as constrained or inaccessible RNA molecules. BNA monomers can contain a five-membered, six-membered or even a seven-membered bridged structure with a "fixed" C3'-endo sugar puckering. The bridge is synthetically incorporated at the 2', 4'-position of the ribose to afford a 2', 4'-BNA monomer. The monomers can be incorporated into oligonucleotide polymeric structures using standard phosphoamidite chemistry. BNAs are structurally rigid oligo-nucleotides with increased binding affinities and stability.

Anti-miRNA oligonucleotides have many uses in cellular mechanics. These synthetically designed molecules are used to neutralize microRNA (miRNA) function in cells for desired responses. miRNA are complementary sequences to mRNA that are involved in the cleavage of RNA or the suppression of the translation. By controlling the miRNA that regulate mRNAs in cells, AMOs can be used as further regulation as well as for therapeutic treatment for certain cellular disorders. This regulation can occur through a steric blocking mechanism as well as hybridization to miRNA. These interactions, within the body between miRNA and AMOs, can be for therapeutics in disorders in which over/under expression occurs or aberrations in miRNA lead to coding issues. Some of the miRNA linked disorders that are encountered in the humans include cancers, muscular diseases, autoimmune disorders, and viruses. In order to determine the functionality of certain AMOs, the AMO/miRNA binding expression must be measured against the expressions of the isolated miRNA. The direct detection of differing levels of genetic expression allow the relationship between AMOs and miRNAs to be shown. This can be detected through luciferase activity. Understanding the miRNA sequences involved in these diseases can allow us to use anti miRNA Oligonucleotides to disrupt pathways that lead to the under/over expression of proteins of cells that can cause symptoms for these diseases.

A hybridization assay comprises any form of quantifiable hybridization i.e. the quantitative annealing of two complementary strands of nucleic acids, known as nucleic acid hybridization.

Miravirsen Medication

Miravirsen is an experimental drug for the treatment of hepatitis C, being developed by Santaris Pharma. As of 2017 it was in Phase II clinical trials.

Gapmers are short DNA antisense oligonucleotide structures with RNA-like segments on both sides of the sequence. These linear pieces of genetic information are designed to hybridize to a target piece of RNA and silence the gene through the induction of RNase H cleavage. Binding of the gapmer to the target has a higher affinity due to the modified RNA flanking regions, as well as resistance to degradation by nucleases. Gapmers are currently being developed as therapeutics for a variety of cancers, viruses, and other chronic genetic disorders.

References

  1. ↑ Elayadi, Anissa N.; Braasch, Dwaine A.; Corey, David R. (August 2002). "Implications of High-Affinity Hybridization by Locked Nucleic Acid Oligomers for Inhibition of Human Telomerase †". Biochemistry. 41 (31): 9973–9981. doi:10.1021/bi025907j. ISSN   0006-2960. PMID   12146961.
  2. ↑ Kurreck, J. (2002-05-01). "Design of antisense oligonucleotides stabilized by locked nucleic acids". Nucleic Acids Research. 30 (9): 1911–1918. doi:10.1093/nar/30.9.1911. PMC   113840 . PMID   11972327.
  3. ↑ Frieden, M. (2003-11-01). "Expanding the design horizon of antisense oligonucleotides with alpha-L-LNA". Nucleic Acids Research. 31 (21): 6365–6372. doi:10.1093/nar/gkg820. ISSN   1362-4962. PMC   275462 . PMID   14576324.
  4. ↑ Frieden, Miriam; Hansen, Henrik F.; Koch, Troels (October 2003). "Nuclease Stability of LNA Oligonucleotides and LNA-DNA Chimeras". Nucleosides, Nucleotides and Nucleic Acids. 22 (5–8): 1041–1043. doi:10.1081/NCN-120022731. ISSN   1525-7770. PMID   14565339. S2CID   10631717.
  5. ↑ Morita, K.; Hasegawa, C.; Kaneko, M.; Tsutsumi, S.; Sone, J.; Ishikawa, T.; Imanishi, T.; Koizumi, M. (2001-11-01). "2'-O, 4'-C-Ethylene-bridged nucleic acids (ENA) with nuclease-resistance and high affnity for RNA". Nucleic Acids Symposium Series. 1 (1): 241–242. doi: 10.1093/nass/1.1.241 . ISSN   0261-3166. PMID   12836354.
  6. ↑ Veedu, Rakesh; Wengel, Jesper (2011). Medicinal Chemistry of Nucleic Acids. John Wiley & Sons, Inc. pp. 335–337. ISBN   978-0470596685.
  7. 1 2 Obika, Satoshi; Nanbu, Daishu; Hari, Yoshiyuki; Morio, Ken-ichiro; In, Yasuko; Ishida, Toshimasa; Imanishi, Takeshi (1997-12-15). "Synthesis of 2′-O,4′-C-methyleneuridine and -cytidine. Novel bicyclic nucleosides having a fixed C3, -endo sugar puckering". Tetrahedron Letters. 38 (50): 8735–8738. doi:10.1016/S0040-4039(97)10322-7. ISSN   0040-4039.
  8. 1 2 3 Orum, Miriam Frieden and Henrik (2008-03-31). "Locked Nucleic Acid Holds Promise in the Treatment of Cancer". Current Pharmaceutical Design. 14 (11): 1138–1142. doi:10.2174/138161208784246234. PMID   18473860 . Retrieved 2020-10-06.
  9. ↑ Zamecnik, P. C.; Stephenson, M. L. (1978-01-01). "Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide". Proceedings of the National Academy of Sciences. 75 (1): 280–284. Bibcode:1978PNAS...75..280Z. doi: 10.1073/pnas.75.1.280 . ISSN   0027-8424. PMC   411230 . PMID   75545.
  10. ↑ Koshkin, Alexei A.; Fensholdt, Jef; Pfundheller, Henrik M.; Lomholt, Christian (2001-12-01). "A Simplified and Efficient Route to 2'-O, 4'-C-Methylene-Linked Bicyclic Ribonucleosides (Locked Nucleic Acid)". The Journal of Organic Chemistry. 66 (25): 8504–8512. doi:10.1021/jo010732p. ISSN   0022-3263. PMID   11735531.
  11. 1 2 Veedu, Rakesh N.; Vester, Birte; Wengel, Jesper (2007-03-26). "Enzymatic Incorporation of LNA Nucleotides into DNA Strands". ChemBioChem. 8 (5): 490–492. doi:10.1002/cbic.200600501. PMID   17315250. S2CID   10206060.
  12. ↑ Breaker, R. R.; Joyce, G. F. (December 1994). "A DNA enzyme that cleaves RNA". Chemistry & Biology. 1 (4): 223–229. doi:10.1016/1074-5521(94)90014-0. ISSN   1074-5521. PMID   9383394.
  13. ↑ Vester, Birte; Lundberg, Lars Bo; Sørensen, Mads D.; Babu, B. Ravindra; Douthwaite, Stephen; Wengel, Jesper (November 2002). "LNAzymes: Incorporation of LNA-Type Monomers into DNAzymes Markedly Increases RNA Cleavage". Journal of the American Chemical Society. 124 (46): 13682–13683. doi:10.1021/ja0276220. ISSN   0002-7863. PMID   12431091.
  14. ↑ Schubert, Steffen; Fürste, Jens P; Werk, Denise; Grunert, Hans-Peter; Zeichhardt, Heinz; Erdmann, Volker A; Kurreck, Jens (May 2004). "Gaining Target Access for Deoxyribozymes". Journal of Molecular Biology. 339 (2): 355–363. doi:10.1016/j.jmb.2004.03.064. PMID   15136038.
  15. 1 2 Petersen M, Wengel J (February 2003). "LNA: a versatile tool for therapeutics and genomics". Trends in Biotechnology. 21 (2): 74–81. doi:10.1016/S0167-7799(02)00038-0. PMID   12573856.
  16. ↑ Dürig, J.; Dührsen, U.; Klein-Hitpass, L.; Worm, J.; Hansen, J. B. Rode; Ørum, H.; Wissenbach, M. (April 2011). "The novel antisense Bcl-2 inhibitor SPC2996 causes rapid leukemic cell clearance and immune activation in chronic lymphocytic leukemia". Leukemia. 25 (4): 638–647. doi: 10.1038/leu.2010.322 . ISSN   1476-5551. PMID   21358717.
  17. ↑ Gebert, Luca F. R.; Rebhan, Mario A. E.; Crivelli, Silvia E. M.; Denzler, Rémy; Stoffel, Markus; Hall, Jonathan (2014-01-01). "Miravirsen (SPC3649) can inhibit the biogenesis of miR-122". Nucleic Acids Research. 42 (1): 609–621. doi:10.1093/nar/gkt852. ISSN   0305-1048. PMC   3874169 . PMID   24068553.
  18. ↑ Bonneau, E.; Neveu, B.; Kostantin, E.; Tsongalis, G.J.; De Guire, V. (2019-06-24). "How close are miRNAs from clinical practice? A perspective on the diagnostic and therapeutic market". EJIFCC. 30 (2): 114–127. ISSN   1650-3414. PMC   6599191 . PMID   31263388.
  19. ↑ Bonetta L (2005). "Prime time for real-time PCR". Nat. Methods. 2 (4): 305–312. doi: 10.1038/nmeth0405-305 . S2CID   17711047.
  20. 1 2 3 Kubota, Kengo; Ohashi, Akiyoshi; Imachi, Hiroyuki; Harada, Hideki (August 2006). "Improved in situ hybridization efficiency with locked-nucleic-acid-incorporated DNA probes". Applied and Environmental Microbiology. 72 (8): 5311–5317. doi:10.1128/AEM.03039-05. ISSN   0099-2240. PMC   1538721 . PMID   16885281.
  21. ↑ van Ravesteyn, TW; Dekker, M; Fish, A; Sixma, TK; Wolters, A; Dekker, RJ; Te Riele, HP (12 April 2016). "LNA modification of single-stranded DNA oligonucleotides allows subtle gene modification in mismatch-repair-proficient cells". Proceedings of the National Academy of Sciences of the United States of America. 113 (15): 4122–7. doi: 10.1073/pnas.1513315113 . PMID   26951689.
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