Locked Nucleic Acid® (LNA®)

LNA is a novel type of nucleic acid analog that contains a 2'-O, 4'-C methylene bridge (Figure 1). This bridge–locked in the 3'-endo conformation–restricts the flexibility of the ribofuranose ring and locks the structure into a rigid bicyclic formation. This confers enhanced assay performance and an increased breadth of applications.

LNA and DNA monomer

 

Figure 1. Structure of LNA and native-state DNA monomers.

LNA qPCR probes are soluble in water and standard buffers as well as follow Watson-Crick base-pairing rules1.

Benefits

When incorporated into qPCR probes, LNA offers several benefits compared to native-state DNA bases only, including the following:

  • Increased thermal stability and hybridization specificity
  • More accurate gene quantification and allelic discrimination
  • Easier and more flexible probe designs for problematic target sequences

Increased Thermal Stability and Hybridization Specificity

Incorporating LNA into a qPCR probe increases thermal duplex stability2 and improves the specificity of probe hybridization to its target sequence3. This reduces background fluorescence from spurious binding, which increases the signal-to-noise ratio. In addition, the improved hybridization of the probe to its target may increase the melting temperature (Tm) by up to 8 °C per LNA monomer substitution in medium salt conditions compared to a native-state DNA qPCR probe4 (Table 1). This increase in hybridization creates a significant broadening in the scope of assay conditions and allows for more successful multiplexing5.

Table 1. Increasing the number of LNA bases in a qPCR probe increases the Tm.
 

Probe Sequence
LNA Bases
Tm*
ΔTm
ΔTm/LNA
GTGATATGC
0
29 °C
 

GTGATATGC
3
44 °C
15 °C
+5 °C
GTGATATGC
9
64 °C
35 °C
+3.9 °C

*TM of duplex between probe and its complementary sequence.
The bolded and underlined bases denote LNA bases.

More Accurate Gene Quantification and Allelic Discrimination

The ability of qPCR probes to discriminate between alleles via SNPs is greatly enhanced by the incorporation of LNA bases6-8 (Figure 2). The presence of a single base mismatch has a greater destabilizing effect on the duplex formation between an LNA qPCR probe and its target than with a native-state DNA qPCR probe. Shorter qPCR probes incorporating LNA bases can be used at the same temperatures as longer native-state DNA qPCR probes.

 

LNA Dual-Labeled Probes discriminate better than DNA Dual-Labeled Probes in SNP genotyping analysis9


Figure 2. LNA Dual-Labeled Probes discriminate better than DNA Dual-Labeled Probes in SNP genotyping analysis9. Pink) mutant DNA analysis with LNA mutant probe (16mer with 3 LNA bases). Green) mutant DNA analysis with native-state DNA mutant probe (25mer). Red) wild-type DNA with native-state DNA mutant probe (25mer). Purple) wild-type DNA with LNA mutant Probe (16mer with 3 LNA bases).

Easier and More Flexible Probe Designs for Problematic Target Sequences

Due to LNA’s improved hybridization characteristics with accompanied increase in Tm, LNA qPCR probes can be synthesized to be shorter, which overcomes certain design limitations that occur with native-state DNA qPCR probes. Specifically, LNA qPCR probes can be designed to address traditionally problematic target sequences, such as AT- or GC-rich regions. For example, AT-rich native-state DNA qPCR probes often need to be over 30 bases long (sometimes over 40 bases) to satisfy amplicon design guidelines but may still perform poorly. With LNA qPCR probes, the selective placement of LNA bases facilitates the optimal design of highly-specific, shorter qPCR probes that perform well, even at lengths of only 13 to 20 bases. Also, the design of qPCR probes for targeting difficult SNPs, such as the relatively stable G:T mismatch, is greatly facilitated by LNA.

Additional Benefit

LNA qPCR probes are compatible with all real-time thermocyclers and end-point analytical detection instruments. No specialized instruments are necessary.

Applications

LNA can be incorporated into all available qPCR detection chemistries, including:

  • Dual-Labeled Probes
  • Molecular Beacons
  • LightCycler® Probes
  • Scorpions® Probes

And, is useful for the following applications:

  • SNP detection
  • Allele discrimination
  • Pathogen detection
  • Multiplexing
  • Viral load quantification
  • Gene expression analysis
  • Gene copy determination

Conclusion

LNA increases the performance of qPCR probes for many applications. Both online and consultative design options are available. If additional help is needed, please consult our technical services group at oligotechserv@sial.com.

Materials

     

References

  1. Egli M, Minasov G, Teplova M, Kumar R, & Wengel J (2001). X-ray crystal structure of a locked nucleic acid (LNA) duplex composed of a palindromic 10-mer DNA strand containing one LNA thymine monomer. Chem Commun, 7, 651-652.
  2. Braasch D, Jensen S, Liu Y, Kaur K, Arar K, White M & Corey D (2003). RNA interference in mammalian cells by chemically-modified RNA. Biochemistry, 42, 7967-7975.
  3. Latorra D, Arar K, & Hurley JM (2003). Design considerations and effects of LNA in PCR primers. Mol Cell Probes, 5, 253-259.
  4. Christensen U, Jacobsen N, Rajwanshi V, Wengel J, & Koch T (2001). Stopped-flow kinetics of Locked Nucleic Acid (LNA)-oligonucleotide duplex formation: studies of LNA-DNA and DNA-DNA interactions. Biochem J, 354, 481-484.
  5. Latorra D, Hopkins D, Campbell K, & Hurley J (2003). Multiplex allele-specific PCR with optimized locked nucleic acid primers. BioTechniques, 34, 1150-1158.
  6. Latorra D, Campbell K, Wolter A, & Hurley J (2003). Enhanced allele-specific PCR discrimination in SNP genotyping using 3’ locked nucleic acid (LNA) primers. Hum Mutat, 22, 79-85.
  7. Mouritzen P, Nielsen A, Pfundheller H, Choleva Y, Kongsbak L, & Moller S (2003).Single nucleotide polymorphism genotyping using locked nucleic acid (LNA). Expert Rev Mol Diagn, 3, 27-28.
  8. Simeonov A & Nikiforov T (2002). Single nucleotide polymorphism genotyping using short, fluorescently labeled locked nucleic acid (LNA) probes and fluorescence polarization detection. Nucleic Acids Res, 30, e91
  9. Ugozzoli L, Latorra D, Pucket R, Arar K, & Hamby K (2004). Real-time genotyping with oligonucleotide probes containing locked nucleic acids. Anal Biochem, 324, 143-152. Erratum: Anal Biochem, 328, 244.