2026-01-19 Posted by TideChem view:86
Locked Nucleic Acid (LNA) phosphoramidites are chemically modified nucleotide building blocks designed to significantly improve the stability and binding performance of oligonucleotides. Their defining feature is a methylene bridge that locks the ribose ring into a fixed conformation, which enhances resistance to nuclease degradation, increases target affinity, and extends in vivo persistence.
LNA phosphoramidites are fully compatible with standard solid-phase oligonucleotide synthesis, making them widely applicable in therapeutics, diagnostics, and molecular biology research. This guide outlines how LNA phosphoramidites work, where they are commonly used, and key practical considerations for synthesis, based on published studies and routine laboratory experience from 2019–2023.
The locked ribose conformation limits enzymatic access to the phosphodiester backbone. Oligonucleotides containing 20–50% LNA residues typically show 10–100× longer serum stability compared with unmodified DNA or RNA.
LNA enforces a C3′-endo–like geometry that improves base stacking and hydrogen bonding. Each incorporated LNA residue increases duplex melting temperature (Tm) by approximately 2–4 °C, allowing strong and specific binding even for short oligonucleotides (10–15 nt).
Together, these properties make LNA-modified oligonucleotides well suited for applications requiring both precision and durability.
Antisense Oligonucleotides (ASOs):
LNA phosphoramidites are used in clinically approved antisense drugs to enhance metabolic stability and reduce dosing frequency. Combining LNA with phosphorothioate (PS) linkages further improves bioavailability.
siRNA and miRNA Therapies:
Selective LNA modification of guide strands protects against RNase degradation and can improve gene-silencing efficiency, particularly in liver, ocular, and neurological indications.
FISH and qPCR Probes:
LNA-modified probes enable detection of low-abundance targets, such as viral RNA or cancer biomarkers. Higher Tm values allow more stringent hybridization conditions, reducing background signals.
CRISPR-Based Diagnostics:
LNA-modified guide RNAs improve stability in CRISPR–Cas13-based detection assays, supporting rapid and sensitive nucleic acid testing.
RNA Structure and Function Studies:
LNA substitutions stabilize short RNA motifs, hairpins, and aptamers for NMR, X-ray crystallography, and biophysical assays.
Gene Editing Controls:
LNA blocking oligonucleotides are used to suppress off-target binding, improving CRISPR–Cas9 editing specificity.
Modification Ratio:
A content of 20–50% LNA residues generally provides the best balance between stability, solubility, and specificity. Excessive modification (>70%) may cause aggregation or poor solubility.
Coupling Conditions:
Use mild detritylation conditions (e.g., 3% DCA in DCM) and extend coupling times to 15–20 minutes to ensure efficient incorporation.
Compatibility:
LNA phosphoramidites are compatible with common modifications such as PS linkages, 2′-O-methyl groups, and fluorescent labels. Place modifications outside critical binding regions when possible.
Storage:
Store LNA phosphoramidites under inert gas at −80 °C for long-term storage or −20 °C for short-term use. Strict moisture control is essential.
Typically 20–50% LNA combined with phosphorothioate linkages for balanced stability and solubility.
Yes. Standard DNA/RNA synthesizers can be used with slightly extended coupling times.
Sealed under argon or nitrogen at −80 °C (long-term) or −20 °C (short-term), protected from moisture.
Yes. Their nuclease resistance and low immunogenicity support both in vitro and in vivo use.
LNA phosphoramidites offer an effective solution to the inherent instability of natural nucleic acids by enhancing both binding affinity and enzymatic resistance. When incorporated thoughtfully—using appropriate modification ratios and optimized synthesis conditions—they enable high-performance oligonucleotides for therapeutic, diagnostic, and research applications.