Locked Nucleic Acids^® (LNA^®) FAQ

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What are locked nucleic acids (LNA)?
What is the difference between dual-labeled fluorescent probes (DLFPs) and LNA probes?
What are the advantages of using LNA probes?
Can I use LNA to raise the Tm of my probes?
Does Sigma design LNA probes?
How do you design LNA probes?
Can you design LNA probes suitable for multiplex assays?
Can LNA probes replace minor groove binding (MGB) probes?
Can LNA be incorporated in DNA primers? Can Sigma provide design for DNA primers with LNA?
Do I need to alter my experimental set up and/or data collection if using probes containing LNA?

What are locked nucleic acids (LNA)?
Locked nucleic acids are RNA monomers with a modified backbone (Exiqon proprietary technology). The sugar phosphate backbone has a 2’-O-4’-C methylene bridge. The bridge increases the monomer’s thermal stability, reduces its flexibility, and increases the hybridization interactions of the base. A comparison of LNA and DNA is shown below:

What is the difference between dual-labeled fluorescent probes (DLFPs) and LNA probes?
DLFPs contain DNA monomers and are generally 25-35 bases in length. LNA probes contain a mixture of DNA and LNA monomers and are generally 14-17 bases in length.

What are the advantages of using LNA probes?
When used with standard bases (A,C,G,T, or U), probes synthesized using LNA have greater thermal stability when compared to conventional DNA or RNA and form a stronger bond with the complementary sequence. The introduction of LNA chemistry into a qPCR probe may result in an increase in the duplex melting temperature (Tm) of up to 8 °C per LNA monomer substitution. It is possible to optimize the Tm level and the hybridization specificity through specific placement of the LNA base(s) in the probe design as shown below.

*Tm of duplex between probe and its complementary sequence.
Note: The bolded and underlined bases denote LNA base.

LNA probes have a more rigid structure and hybridization strength that confers greater sensitivity and discrimination for the gene target. This makes them an excellent tool for SNP analysis/ allelic discrimination, as they can discriminate between highly homologous targets with a single base difference.

LNA monomers have higher thermal stability enabling you to use much shorter probes (14-17 bases) in your assay. In qPCR assays, the probe needs to have an approximately 5-10 ºC greater melting temperature (Tm) than the primers. This ensures that during the annealing phase of PCR, the probe will bind to the target before the primers, and is in place to be hydrolyzed by the Taq polymerase and emit a fluorescent signal. The higher thermal stability of LNA bases means that short probes will have a high melting temperature and anneal to the target before the primers. The design of shorter probes is a valuable tool when working with complex targets and very small gene fragments, for example viral genes, where longer dual labeled probes are unsuitable.

Can I use LNA to raise the Tm of my probes?
A key characteristic of LNA and the addition of a 2’-O-4’-C methylene bridge is an increase in thermal stability. Incorporation of LNA bases into your oligo will increase the oligos Tm by 3-5 degrees depending upon which bases are used and where they are placed (see table).

Does Sigma design LNA probes?
Sigma offers a free qPCR primer and probe design service including advice on assay design, optimization and troubleshooting. We use Beacon Designer (Premier Biosoft) and routinely provide design for primers and a variety of probes including: dual labeled probes, LNA probes, Molecular Beacons, Scorpions™ and LightCycler^® probes. Design requests can be submitted at sigma.com/designmyprobe.

How do you design LNA probes?
LNA is a recommended oligonulceotide modification for SNP/allelic discrimination assays and also to increase the Tm in a short probe region. We generally recommend LNA bases to be inserted every 2-3 bases along the probe, avoiding the 5’ and 3’ ends and highly folded regions.

For SNP assays, we recommend positioning the SNP in the center of the probe and inserting a single or triplet of LNA bases at this position to improve base discrimination.

Large clusters of LNA bases at the 5’ and 3’ regions may cause the probe to bind too tightly to the target. This can cause the Taq polymerase to be knocked off the target, or prevent it from cleaving the probe and releasing a fluorescent signal. It is also important to avoid inserting LNA bases in highly folded regions of the probe where dimers or loops may form. LNA bases have stronger hybridization interactions compared to DNA monomers, therefore the positioning of LNA bases in folded regions may lead to the formation of strong probe dimers or loop structures. The kinetics of forming these structures may be stronger than binding to the gene target, and the probe may effectively quench itself out of the reaction.

For advice on LNA probe design please contact our technical team or visit sigma.com/designmyprobe.

Can you design LNA probes suitable for multiplex assays?
Sigma uses Beacon Designer software (Premier Biosoft) for primer and probe design. Beacon Designer software has a “LNA probe design” and a “LNA multiplex design” function which has been validated and used for design of multiplex assays for both DLFP and LNA probes. Sigma can design assays with up to four different gene targets in a multiplex reaction.

Can LNA probes replace minor groove binding (MGB) probes?
MGB probes are licensed to Applied Biosystems and are not supplied by Sigma. MGB probes are short (12-14 bases) and are designed to sit in the minor groove of the gene target. Sigma can use LNA to increase the probe Tm. However this is not always feasible and we prefer to design the probe de novo when possible, as the position of the MGB probe is not always suitable for LNA probe design.

Can LNA be incorporated in DNA primers? Can Sigma provide design for DNA primers with LNA?
LNAs are modified RNA monomers and can therefore be incorporated into all types of synthetic oligonucleotides. However, Sigma only offers LNA in probes for qPCR. We are unable to offer or design other types of LNA modified oligonucleotides.

Do I need to alter my experimental set up and/or data collection if using probes containing LNA?
No. DLFPs and LNA probes are hydrolysis probes. The experimental set up and data collection will be the same for both probe types. Data is collected during the annealing/extension phase of each cycle when the Taq polymerase encounters the probe on the target and its exonuclease activity cleaves the probe.

Are there any special resuspension, storage and/or handling requirements for LNA probes?
No. LNA probes should be resuspended, stored and handled in a similar fashion as dual labeled probes. Storage and handling guidelines can be found in our custom oligonucleotide technical resources section on the website, sigma.com/oligos.

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