![LNASchem](https://upload.wikimedia.org/wikipedia/commons/0/07/LNASchem.svg)
A locked nucleic acid (LNA), also known as bridged nucleic acid (BNA), and often referred to as inaccessible RNA, is a modified
RNA
Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...
nucleotide
Nucleotides are organic molecules consisting of a nucleoside and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecules wi ...
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. LNA also offers improved specificity and affinity in base-pairing as a monomer or a constituent of an oligonucleotide. LNA nucleotides can be mixed with DNA or RNA residues in a oligonucleotide.
Synthesis
Obika et al. were the first to chemically synthesize LNA in 1997,
independently followed by Jesper Wengel's group in 1998.
This became possible after Zamecnick and Stephenson laid the groundwork on the possibility of
oligonucleotide
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 c ...
s being great agents for controlling gene expression in 1978. 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.
In this approach,
uridine
Uridine (symbol U or Urd) is a glycosylated pyrimidine analog containing uracil attached to a ribose ring (or more specifically, a ribofuranose) via a β-N1-glycosidic bond. The analog is one of the five standard nucleosides which make up nuclei ...
(or any readily available RNA
nucleoside
Nucleosides are glycosylamines that can be thought of as nucleotides without a phosphate group. A nucleoside consists simply of a nucleobase (also termed a nitrogenous base) and a five-carbon sugar (ribose or 2'-deoxyribose) whereas a nucleotide ...
) can be used as the starting material. The convergent strategy requires the synthesis of a sugar intermediate which serves a
glycosyl
A glycosyl group is a univalent free radical or substituent structure obtained by removing the hemiacetal hydroxyl group from the cyclic form of a monosaccharide and, by extension, of a lower oligosaccharide.
Glycosyl also reacts with inorganic a ...
donor necessary for coupling with
nucleobase
Nucleobases, also known as ''nitrogenous bases'' or often simply ''bases'', are nitrogen-containing biological compounds that form nucleosides, which, in turn, are components of nucleotides, with all of these monomers constituting the basic b ...
s. Commonly,
D-glucose
Glucose is a simple sugar with the molecular formula . Glucose is overall the most abundant monosaccharide, a subcategory of carbohydrates. Glucose is mainly made by plants and most algae during photosynthesis from water and carbon dioxide, usin ...
is used to produce the sugar intermediate which is subsequently reacted with nucleobases using a modified Vorbrügen procedure allowing for stereoselective coupling.
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.
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
''Pfu'' DNA polymerase is an enzyme found in the hyperthermophilic archaeon ''Pyrococcus furiosus'', where it functions to copy the organism's DNA during cell division. In the laboratory setting, ''Pfu'' is used to amplify DNA in the polymeras ...
, efficiently incorporates LNA into DNA.
Properties
LNA offers enhanced biostability compared to biological
nucleic acid
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 cl ...
s. LNA modified oligonucleotides have demonstrated improved thermodynamics in hybridization to
RNA
Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...
,
ssDNA, and
dsDNA.
[
]
Applications
LNAzymes
DNAzyme
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, ...
s can be modified to include LNA residues, producing LNAzymes (LNA-modified DNAzymes). These modified oligonucleotides, like their DNAzyme relatives, are generally endonuclease
Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. Some, such as deoxyribonuclease I, cut DNA relatively nonspecifically (without regard to sequence), while many, typically called restriction endonucleases ...
s that bind to specific RNA target sequences and cleave the phosphodiester bond
In chemistry, a phosphodiester bond occurs when exactly two of the hydroxyl groups () in phosphoric acid react with hydroxyl groups on other molecules to form two ester bonds. The "bond" involves this linkage . Discussion of phosphodiesters is ...
that exists between the nucleotides. However, they demonstrate more efficient cleavage of phosphodiester bond
In chemistry, a phosphodiester bond occurs when exactly two of the hydroxyl groups () in phosphoric acid react with hydroxyl groups on other molecules to form two ester bonds. The "bond" involves this linkage . Discussion of phosphodiesters is ...
s compared to their unmodified counterparts. Modification of the substrate recognition arms of DNAzyme
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, ...
s with LNA monomer
In chemistry, a monomer ( ; ''mono-'', "one" + '' -mer'', "part") is a molecule that can react together with other monomer molecules to form a larger polymer chain or three-dimensional network in a process called polymerization.
Classification
Mo ...
s yields a LNAzyme which recognizes coxsackievirus
Coxsackieviruses are a few related enteroviruses that belong to the ''Picornaviridae'' family of viral envelope, nonenveloped, linear, positive-sense single-stranded RNA viruses, as well as its genus ''Enterovirus'', which also includes poliovi ...
A21 (CAV-21) and cleaves its RNA
Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...
target sequence similar to one in the 5' untranslated region
The 5′ untranslated region (also known as 5′ UTR, leader sequence, transcript leader, or leader RNA) is the region of a messenger RNA (mRNA) that is directly Upstream and downstream (DNA), upstream from the initiation codon. This region is im ...
(5' UTR) of the human rhinovirus
The rhinovirus (from the grc, ῥίς, rhis "nose", , romanized: "of the nose", and the la, vīrus) is the most common viral infectious agent in humans and is the predominant cause of the common cold. Rhinovirus infection proliferates in tem ...
-14 (HRV-14); a sequence unrecognized by unmodified DNAzymes.
Therapeutics
Using LNA based oligonucleotides
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 c ...
therapeutically is an emerging field in biotechnology
Biotechnology is the integration of natural sciences and engineering sciences in order to achieve the application of organisms, cells, parts thereof and molecular analogues for products and services. The term ''biotechnology'' was first used b ...
. 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.
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
In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of Protein biosynthesis, synthesizing a protein.
mRNA is ...
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.
LNA has also been applied to Miravirsen, an experimental therapeutic intended for the treatment of Hepatitis C
Hepatitis C is an infectious disease caused by the hepatitis C virus (HCV) that primarily affects the liver; it is a type of viral hepatitis. During the initial infection people often have mild or no symptoms. Occasionally a fever, dark urine, a ...
, constituting a 15-nucleotide phosphorothioate sequence with binding specificity for MiR-122
miR-122 is a miRNA that is conserved among vertebrate species. miR-122 is not present in invertebrates, and no close paralogs of miR-122 have been detected. miR-122 is highly expressed in the liver, where it has been implicated as a regulator of ...
(a miRNA
MicroRNA (miRNA) are small, single-stranded, non-coding RNA molecules containing 21 to 23 nucleotides. Found in plants, animals and some viruses, miRNAs are involved in RNA silencing and post-transcriptional regulation of gene expression. miRN ...
expressed in hepatocyte
A hepatocyte is a cell of the main parenchymal tissue of the liver. Hepatocytes make up 80% of the liver's mass.
These cells are involved in:
* Protein synthesis
* Protein storage
* Transformation of carbohydrates
* Synthesis of cholesterol, ...
s).
Detection and diagnosis
Allele-specific PCR using LNA allows for the design of shorter primers, without compromising binding specificity.
LNA has been incorporated in fluorescence ''in situ'' hybridization (FISH). 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
Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...
. The improved efficiency of LNA-incorporated FISH has resulted in FISH analysis of the human chromosome, several types of non-human cells, and microarrays.
LNA genotyping assays have been conducted as well, specifically to detect a mutation in apolipoprotein B
Apolipoprotein B (ApoB) is a protein that in humans is encoded by the gene.
Function
Apolipoprotein B is the primary apolipoprotein of chylomicrons, VLDL, Lp(a), IDL, and LDL particles (LDL—commonly known as "bad cholesterol" when in refer ...
.
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
SNP genotyping is the measurement of genetic variations of single nucleotide polymorphisms (SNPs) between members of a species. It is a form of genotyping, which is the measurement of more general genetic variation. SNPs are one of the most common ...
assay.
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
DNA mismatch repair (MMR) is a system for recognizing and repairing erroneous insertion, deletion, and mis-incorporation of bases that can arise during DNA replication and recombination, as well as repairing some forms of DNA damage.
Mismatch ...
due to the higher thermodynamic stability it has.
References
{{DEFAULTSORT:Locked Nucleic Acid
Nucleic acids