Locked Nucleic Acid (LNA®) was first described by Wengel and co-workers in 1998 as a novel class of conformationally restricted oligonucleotide analogues. LNA® is a bicyclic nucleic acid where a ribonucleoside is linked between the 2’-oxygen and the 4’-carbon atoms with a methylene unit.
By changing the conformation of the helix and by increasing the stability of the duplex, the integration of LNA® bases into your oligo sequence opens new perspectives to your DNA affinity based studies. For instance, LNA® may be used to improve techniques requiring high affinity probes as specific as possible like SNP detection, expression profiling, and in situ hybridization.
What does it look like?
LNA® is a bicyclic RNA analogue, in which the ribose moiety in the sugar-phosphate backbone is structurally constrained by a methylene bridge between the 2’-oxygen and the 4’-carbon atoms (Obika et al. 1997, Koskhin et al. 1998, Singh et al. 1998).
The pre-organized conformation of the LNA® nucleoside was predicted to be a N-type sugar puckering (Fig.1), characteristic for A-type double helices, such as RNA-RNA duplexes. This assumption has been confirmed by NMR solution studies and X-ray crystallographic analysis. The LNA® oligonucleotide conformational structure, examining both sugar puckering and oligonucleotide backbone, has been determined by two-dimensional NMR analysis. The preliminary LNA® nucleoside spectra demonstrated the fixed N-type conformation of LNA® (Koskhin et al. 1998, Singh et al. 1998). Subsequent NMR studies have analyzed the structure of LNA® oligonucleotides, either as single stranded oligonucleotides or hybridized to complementary DNA and RNA (Nielsen et al. 1999, Bondensgaard et al. 2000, Nielsen et al. 2000, Petersen et al. 2000, Petersen et al. 2002). The spectra confirmed the locked N-type conformation of the LNA® sugar pucker, but also revealed that LNA® monomers are able to twist the neighbouring, unmodified nucleotides from an S-type towards an N-type conformation in DNA/LNA® mixmer oligos and LNA®-containing duplexes.
The fixed N-type (3’-endo) conformation of the LNA® nucleoside, together with enhanced stacking of the nucleobases results in higher thermal stability of LNA®-containing duplexes.
The structural consequence
The integration of LNA® bases into probes changes the conformation of the duplex when the annealing with DNA bases occurs. The integration of LNA® moieties on every third position changes the structure of the double helix from the B to the A type. This conformation allows a much better stacking and then a higher stability.
An increased Tm: the direct advantage
By increasing the stability of the duplex, the integration of LNA® monomers into the oligonucleotide sequence consequently increases the Tm of the duplex.
This characteristic allows a reduction in the size of the probe and, therefore, increases its specificity.
Each LNA® incorporation increases the Tm of the duplex. The following table shows the average Tm increase for DNA or RNA duplex with oligos containing either LNA®, RNA or PNA moieties.
LNA® should be used in any hybridization assay, which requires high specificity and/or reproducibility. LNA® may be used to enhance:
- Real-Time qPCR probes
- in situ hybridization probes
- Primers for single, multiplex and allele specific PCR
- Capture probes for SNP genotyping
- Capture probes for expression analysis
- Probes to monitor exon skipping
- Improvement of SNP discrimination
The LNA® modification perfectly suits to SNP detection
First, the reduction of the size of the probe increases the impact of one mismatch in the stability of the duplex probe/target.
Also, by designing probes with an LNA® moiety in front of the variable position it becomes possible to discriminate very efficiently the allelic variations. The mismatch would avoid the A helix structure stabilisation and then decrease the Tm considerably. This modification not only increases the specificity of the probe but also its power of discrimination.
- Increased affinity
LNA® increases the thermal stability of duplexes due to its RNA-like structure.
LNA®:LNA® duplex formation constitutes the most stable Watson-Crick base pairing system.
- Better Tm modulation
Depending on their position along the sequence, LNA® bases provide a means to reach the desired Tm level without losing specificity.
Introduction of LNA® allows for shorter probes while maintaining the same Tm.
- Increased specificity
LNA® enhances hybridization perfomance relative to native DNA, RNA or phosphorothioate.
LNA® lowers experimental error rates due to better mismatch discrimination.
LNA® improves signal-to-noise ratio.
- Enzyme compatibility
LNA® shows increased resistance to certain exo- and endonucleases thus leading to biostability making this modification perfect for in vivo antisense applications.
DNA-LNA® chimeras readily activate RNase H.
LNA® acts as a substrate for standard molecular biology enzymes: T4 PNK, T4 DNA ligase, DNA polymerases.
LNA® behaves like DNA, so it is easily transferable to DNA-based assays. Moreover LNA® can also be coupled to RNA bases.
LNA® is highly soluble in water.
LNA® complies with almost all oligonucleotide synthesis and analysis methods (QC, purification, etc) and exhibits the same salt dependence as DNA and RNA.
Eurogentec offers a full range of products and services to deliver the LNA® based probes for your application.
All four LNA® bases (LNA®-A, LNA®-T, LNA®-G and 5-Me-LNA®-C) can be mixed with DNA, RNA as well as other nucleic acid analogues using standard phosphoramidite DNA synthesis chemistry. Therefore, LNA® oligonucleotides can be tagged with e.g. aminolinkers, biotin, fluorophores, etc. Thus a very high degree of freedom in the design of primers and probes exists.
The oligonucleotides containing LNA® bases are desalted, deprotected, chromatography purified and controlled by MALDI-TOF Mass Spectrometry.