Locked Nucleic Acid (LNA™) Technology

An LNA™ oligonucleotide offers substantially increased affinity for its complementary strand, compared to traditional DNA or RNA oligonucleotides. This results in unprecedented sensitivity and specificity and makes LNA™ oligonucleotides ideal for the detection of small or highly similar DNA or RNA targets.

The benefits of LNA™ include:

• Significantly increased sensitivity compared to DNA and RNA oligos/probes
• Enables robust detection of all microRNA sequences, regardless of GC content
• Superior detection from challenging clinical samples such as biofluids and FFPE
• Increased target specificity compared to DNA and RNA probes
• Enables detection of single nucleotide mismatches
• Superior discrimination of microRNA families
• High in vivo and in vitro stability
• Enables high potency binding to RNA and DNA
• Superior antisense inhibition of small RNA targets in vivo.

Locked nucleic acids (LNA™) are a class of high-affinity RNA analogs in which the ribose ring is “locked” in the ideal conformation for Watson-Crick binding (Figure 1). As a result, LNA™ oligonucleotides exhibit unprecedented thermal stability when hybridized to a complementary DNA or RNA strand. For each incorporated LNA™ monomer, the melting temperature (Tm) of the duplex increases by 2-8 ºC (Figure 2). In addition, LNA™ oligonucleotides can be made shorter than traditional DNA or RNA oligonucleotides and still retain a high Tm. This is important when the oligonucleotide is used to detect small or highly similar targets.

Since LNA™ oligonucleotides typically consist of a mixture of LNA™ and DNA or RNA, it is possible to optimize the sensitivity and specificity by varying the LNA™ content of the oligonucleotide. Incorporation of LNA™ into oligonucleotides has been shown to improve sensitivity and specificity for many hybridization-based technologies including PCR, microarray and in situ hybridization.

Figure 2
Replace DNA with LNA™ for higher Tm. (Click to learn more)


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LNA™-based oligonucleotides are the best solution for highly sensitive and specific analysis of short RNA and DNA targets.

Tm normalization – robust detection regardless of GC content

The Tm of a nucleotide duplex can be controlled by varying the LNA™ content. This feature can be used to normalize the Tm across a population of short sequences with varying GC-content. For AT-rich nucleotides, which give low melting temperatures, more LNA™ is incorporated into the LNA™ oligonucleotide to raise the Tm of the duplex. This enables the design of LNA™ oligonucleotides with a narrow Tm range which is beneficial in many research applications such as microarray, PCR and other applications where sensitive and specific binding to many different targets must occur under the same conditions simultaneously. The power of Tm-normalization is demonstrated by the comparison of DNA and LNA™ probes for detection of microRNA targets with a range of CG content (Figure 1).

“LNA™-enhanced oligonucleotides can be designed to have a similar affinity towards all types of sequences regardless of the GC-content”

Figure 1
The power of Tm normalization. (Click to learn more)

 

Superior single nucleotide discrimination

Intelligent placement of LNA™ monomers can also ensure excellent discrimination between closely related sequences down to as little as one nucleotide difference. The difference in Tm between a perfectly matched and a mismatched target is described as the delta Tm. Incorporation of LNA™ in oligonucleotides can increase the delta Tm between perfect match and mismatch binding by up to 8 °C. The increase in delta Tm enables better discrimination between closely related sequences such as members of microRNA families.

Broad applicability

The affinity-enhancing effects of LNA™ give LNA™ oligonucleotides strand invasion properties making LNA™ excellent for in vivo applications. Incorporation of LNA™ into oligonucleotides further increases resistance to endo- and exonucleases which leads to high in vitro and in vivo stability.

Since the physical properties (e.g. water solubility) of these sequences are very similar to those of RNA and DNA, conventional experimental protocols can easily be adjusted to their use.

Superior results from challenging clinical samples The increase in sensitivity and specificity of LNA™-enhanced oligonucleotides makes them ideal for challenging applications where the target is present at low levels. For example, LNA™-enhanced PCR primers are superior for quantifying short RNAs in small amounts of biofluids such as serum and plasma1 and LNA™-enhanced capture probes offer excellent sensitivity and signal-to-noise ratios in FFPE samples, where short RNA targets such as microRNAs are present in a background of highly degraded RNA. 1) Jensen et al. BMC Genomics 2011

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LNA™ enables robust detection of all microRNA sequences, regardless of GC content.

The small sizes and widely varying GC-content (5-95 %) of microRNAs make them challenging to analyze using traditional methods. The use of DNA or RNA based technologies for microRNA analysis can introduce high uncertainty and low robustness because the melting temperature (Tm) of the oligonucleotide/microRNA duplex will vary greatly depending on the GC content of the sequences. This is especially problematic in applications such as microarray profiling and high throughput experiments where many microRNA targets are analyzed under the same experimental conditions.

These challenges in microRNA analysis can be overcome by using LNA™-enhanced oligonucleotides. By simply varying the LNA™ content, oligonucleotides with specific duplex melting temperatures can be designed, regardless of the GC-content of the microRNA. Exiqon has used the LNA™ technology to Tm-normalize primers, probes and inhibitors to ensure that they all perform well under the same experimental conditions.

Another challenge of studying microRNAs is the high degree of similarity between the sequences. Some microRNA family members vary by a single nucleotide. LNA™ can be used to enhance the discriminatory power of primers and probes to allow excellent discrimination of closely related microRNA sequences. LNA™ offers significant improvement in sensitivity and specificity and ensures optimal performance for all microRNA targets.

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miRCURY LNA™ products

LNA™ is a powerful tool for nucleic acids research.

The unique characteristics of LNA™ make it a powerful tool, not only for microRNA research but also for detection of low abundance, short or highly similar targets in a number of other applications (Figure 1).

LNA™ has been successfully used to overcome the difficulties of studying very short sequences and has greatly improved, and in many cases enabled, specific and sensitive detection of non-coding RNA and other small RNA molecules. The unique ability of LNA™ oligonucleotides to discriminate between highly similar sequences has further been exploited in a number of applications targeting longer RNA sequences such as mRNA. In addition LNA™ has been successfully used for detection of low abundance nucleic acids and chromosomal DNA.

Figure 1
Proven LNA™ applications. LNA™ is a powerful tool in many applications where standard DNA or RNA oligonucleotides do not have sufficient affinity or specificity. The figure shows an overview of some of the LNA™ applications that have been used for the study of RNA and DNA.



More information on LNA™ (pdf)

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