Locked Nucleic Acid (LNA) Technology

LNA enhancement for highly sensitive and specific analysis of short RNA and DNA targets
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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 highly suited for the detection of small or highly similar DNA or RNA targets.
What are locked nucleic acids (LNA)?
Why use LNA?
Superior single nucleotide discrimination
LNA in miRNA studies
Broad applicability
What are locked nucleic acids (LNA)?
Locked nucleic acids are a class of high-affinity RNA analogs in which the ribose ring is "locked" in the ideal conformation for Watson-Crick binding (see figure Structure of LNA). 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°C (see figure Replace DNA with LNA for higher melting temperature). 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, microarrays and in situ hybridization (ISH).

Why use LNA?
Tm normalization enables 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 microarrays, PCR and other applications in which 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 miRNA targets with a range of CG content (see Figure The power of Tm normalization).

The benefits of LNA include:
  • Significantly increased sensitivity compared to DNA and RNA oligos/probes
  • Robust detection of all miRNA sequences, regardless of GC content
  • Superior detection from challenging samples, such as biofluids and FFPE samples
  • Increased target specificity compared to DNA and RNA probes
  • Enables detection of single nucleotide mismatches
  • Superior discrimination of miRNA 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

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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 the ΔTm. Incorporation of LNA in oligonucleotides can increase the ΔTm between perfect match and mismatch binding by up to 8°C. The increase in ΔTm enables better discrimination between closely related sequences, such as members of miRNA families.

Superior results from challenging samples
The increase in sensitivity and specificity of LNA-enhanced oligonucleotides makes them ideal for challenging applications, in which 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 plasma (1), and LNA-enhanced capture probes offer excellent sensitivity and signal-to-noise ratios in FFPE samples, where short RNA targets, such as miRNAs, are present in a background of highly degraded RNA.

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LNA in miRNA studies
The small sizes and widely varying GC content (5–95%) of miRNAs make them challenging to analyze using traditional methods. The use of DNA- or RNA-based technologies for miRNA analysis can introduce high uncertainty and low robustness, because the Tm of the oligonucleotide/miRNA 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 involving the analysis of many miRNA targets under the same experimental conditions.

These challenges in miRNA 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 miRNA. We have used LNA technology to Tm-normalize primers, probes and inhibitors, to ensure that they all perform well under the same experimental conditions (see figure LNA miRNA inhibitors have high uniform potency).

Another challenge of studying miRNAs is the high degree of similarity between the sequences. Some miRNA 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 miRNA sequences. LNA offers significant improvement in sensitivity and specificity and ensures optimal performance for all miRNA targets.

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Broad applicability
The unique characteristics of LNA make it a powerful tool, not only for miRNA research but also for detection of low-abundance, short or highly similar targets in a number of applications.

miRNA analysis:
  • Real-time, quantitative PCR
  • Microarray analysis
  • In situ hybridization
  • Northern blotting
  • Bead-based applications
  • Inhibition of RNA function

DNA analysis:
  • Real-time, quantitative PCR
  • SNP detection by allele-specific PCR
  • Bead-based applications
  • Chromosomal FISH
  • Comparative genome hybridization
  • Proteomics of isolated chromatin segments (PICh)
  • Antigene inhibition
  • Mutagenesis

ncRNA analysis:
  • Real-time, quantitative PCR
  • Microarray analysis
  • In situ hybridization
  • Northern blotting
  • Fluorescence-activated cell sorting
  • Inhibition of RNA function
  • RNA modification (frame shifting, exon skipping)

mRNA analysis:
  • Real-time, quantitative PCR
  • Microarray analysis
  • In situ hybridization
  • Northern blotting
  • Bead-based applications
  • Fluorescence-activated cell sorting
  • Inhibition of RNA function
  • RNA modification (frame shifting, exon skipping)
  • DNAzymes

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 been further 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.

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 endonucleases 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 for their use.

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References
1. Jensen et al. (2011)  Evaluation of two commercial global miRNA expression profiling platforms for detection of less abundant miRNAs. BMC Genomics. 12:435. doi: 10.1186/1471-2164-12-435.