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Locked nucleic acids and their applications

Increase oligonucleotide hybridization Tm, target specificity, nuclease resistance, and mismatch discrimination

What are locked nucleic acids?

Locked nucleic acids are modified RNA monomers. The “locked” part of their name comes from a methylene bridge bond linking the 2′ oxygen to the 4′ carbon of the RNA pentose ring (Figure 1). The bridge bond fixes the pentose ring in the 3′-endo conformation.

These bases follow Watson-Crick base-pairing rules when mixed with DNA or RNA bases in an oligonucleotide. When incorporated into an oligonucleotide probe, locked nucleic acid monomers impart heightened structural stability, resulting in increased hybridization melting temperature (Tm), both in vitro and in vivo. They also provide significant resistance to nucleases, enzymes that would otherwise degrade the DNA or RNA sequence.

Figure 1. A locked nucleic acid monomer. These modified bases contain a methylene bridge bond (orange) between the 2′ oxygen and the 4′ carbon of the pentose ring.

Manage sequence Tm using locked nucleic acid bases

Because of the afforded increase in Tm, locked nucleic acid qPCR probes can be designed with shorter lengths than standard probes. Shorter probes are more effectively quenched and have a higher signal-to-noise ratio. They are, therefore, more sensitive, providing robust target detection regardless of sequence GC content.

These probes also show greater mismatch discrimination compared to traditional qPCR probes. This improves their ability to distinguish mutations or single nucleotide polymorphisms (SNPs) [1]. By including locked nucleic acid bases, you can design a probe with ΔTm >15°C. By varying the number of these modified bases, you can essentially control the Tm of a nucleotide duplex. Use these probes in methods that use differential hybridization to distinguish polymorphisms.

Other applications of locked nucleic acid probes include transcript variant identification; verification of microbial species; and improved target detection in FFPE tissue, biofluids, and other challenging samples.

Locked nucleic acid oligonucleotides give control over hybridization

Locked nucleic acid oligonucleotides are useful in template switching oligo designs and for strengthening target oligo binding in challenges sequence regions, such as AT-rich areas. As with locked nucleic acid qPCR probes, hybridization Tm can be manipulated by the number of locked nucleic acid bases incorporated.

Improve your hybridization applications with locked nucleic acid technology

IDT provides 2 types of locked nucleic acid products: Custom Affinity Plus DNA & RNA Oligonucleotides, and Affinity Plus qPCR Probes. While Affinity Plus modified sequences provide identical performance to traditional LNA® sequences, PrimeTime Affinity Plus qPCR Probes and custom Affinity Plus DNA & RNA Oligonucleotides are a better value.

*LNA is a registered trademark of Qiagen, Inc. 

Affinity Plus qPCR Probes

Use Affinity Plus qPCR Probes for SNP genotyping, transcript variant identification, and sensitive target detection in challenging samples (FFPE tissue, biofluids).

Affinity Plus DNA & RNA Oligonucleotides

Use Affinity Plus DNA & RNA Oligonucleotides for increased hybridization Tm, stability, and nuclease resistance over standard oligonucleotides.

References

  1. Davalieva K, Kiprijanovska S, et al. (2014) Fast, reliable and low cost user-developed protocol for detection, quantification and genotyping of hepatitis C virus. J Virol Methods, 196:104–112.