Locked nucleic acid

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.

Synthesis

Obika et al. were the first to chemically synthesize LNA in 1997,

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

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