LNA primers and probes
Locked Nucleic Acid is a type of nucleic acid analog that contains a 2'-O, 4'-C methylene bridge.
Figure 1. Structure of Locked Nucleic Acid and native-state DNA monomers.
The “locked” part of their name comes from a methylene bridge bond linking the 2′ oxygen to the 4′ carbon of the RNA pentose ring (Figure 1). The bridge bond fixes the pentose ring in the 3′-endo conformation. In fact, LNA nucleotides are RNA building blocks due to the 2´oxygen on the sugar component in place, however not requiring a 2´OH protection group (like TBDMS, TOM, ACE or TC) as “the lock” provides protection.
LNA building blocks in PCR primers, qPCR probes, and other types of oligonucleotides provide “oligo solubility” in water and standard buffers. And as the modification concerns the Nucleic Acid backbone only, Watson-Crick base-pairing rules remain unaffected.
LNA modified oligonucleotides advantages at a glance
- increased thermal stability and hybridization specificity
- increased Tm for short and AT-rich primers and probes (ΔTm/locked base approx. 2-6°C depending on sequence composition and “neighbourhood” effects)
- Enhanced in-vitro and in-vivo stability due to increased endo- and exonuclease resistance
- Nontoxic, efficient and effective building blocks for incorporation in antisense, m-RNA or other Nucleic Acid based drug development candidates.
... providing for
- efficient mismatch/SNP/allelic discrimination
- earlier Ct values
- higher signal level/signal-to-noise ratio
- facilitated and flexible designs for problematic target sequences – however sequence dependent.
- an excellent supplementation to our Tm/specificity/sensitivity increasing portfolio: ZNA and MGB modified oligonucleotides.
... hence qualifying for
a wide range of Nucleic Acid-based applications like
- PCR/real-time PCR/RT-PCR
- Microarrays/Capture probing
- Gene silencing
- In Situ Hybridization (ISH)
The best way to explore our standard portfolio for LNA modified primers and probes is to log into our Web Order Portal (WOP) and visit the menu items marked with the red NEW suffix. We hope you enjoy the options presented!
LNA probes rely on modified nucleotide chemistry with regard to the sugar component involved, while the organic base component is “unmodified” and thus follows Watson-Crick base-pairing rules when mixed with DNA or RNA bases in an oligonucleotide. When incorporated into an oligonucleotide probe, locked nucleic acid monomers increase structural stability, resulting in a raise of the formed duplex´melting temperature (Tm). Locked Nucleic acids are not recognized by DNA/RNAses as a substrate, hence LNA modified oligonucleotides also display significant resistance to nucleases.
In contrast, ZNA®s are oligonucleotides conjugated with repeated cationic spermine units that decrease electrostatic repulsions with target nucleic acid strands, and greatly improve hybridization properties by enhanced affinity to the complementary target sequence as well as increased stability of the formed duplex at an unprecedented specificity. The “Tm boost” generated by adding ZNA to either end of the oligonucleotide probe is significant and sequence-independent, meaning the core sequence remains “unmodified”.
MGB (Minor Groove Binder), ZNA®, and LNA (Locked Nucleic Acids) are known to increase the Tm of an oligo sequence. MGB (minor groove binder) probes include a minor groove binder (MGB) moiety at the 3’ end that increases the melting temperature (Tm) of the probe and stabilizes the hybridization of the probe DNA to its target sequence. The introduction of the minor groove binder moiety is sequence-independent, too.
In summary, metabion offers all three duplex stability enhancing modifications, and therefore provides greatest flexibility in assay design and choosing the right option for your required application.
The preferred way is order transmission through our Web Order Portal (WOP) for most convenient online shopping.
Alternatively orders can be placed by sending an e-mail with our pre-formatted Excel order file as attachment.
In the section “Dual Labelled Probe type” you can find various 5’ Reporter-3´ Quencer combinations. Select the one of your choice and mark the respective LNA nucleotides in the sequence as (+A), (+C), (+G), and (+T) respectively.
R-Q combinations and yield ranges exceeding our standard portfolio can certainly be inquired.
A hardcopy of the HPLC chromatogram and the mass spectrum are included in addition to the respective Synthesis Report and Delivery Note.
Yes, this is possible. Please mark the respective phosphorothioate bond by an axterix (*).
A fully thioated LNA 5´ATCGAT3´ Hexamer for example shall be depicted as follows:
5´ (+A) *(+T) *(+C) *(+G) *(+A) *(+T) 3´
When designing LNA-containing oligonucleotides, one should follow the basic rules of primer design particularly pay attention to the location and number of LNAs.
For example, a typical 18-mer should contain a maximum of 7–8 LNAs.
Also, try to avoid stretches of more than 4 consecutive LNAs, which would result in very tight hybridization in that region.
Stretches of LNAs are to be avoided close to the 3′ end of an oligonucleotide. LNA will bind very tightly to other LNA residues. Avoid self-complementarity and cross-hybridization to other LNA-containing oligonucleotides.
Finally, be sure to match the Tm of the primers, as usual, keeping in mind that each substitution of a standard nucleotide with an LNA increases the Tm by 2-6°C per LNA.
For novel applications, design guidelines may have to be established empirically by creating your own “best recipe”.
For LNA probes selected from our standard portfolio you can expect a TAT of 5 business days.
A concentration of 0.2 µM of the LNA probe should be fine for most assays, but as usual the optimal concentration should be determined experimentally. Due to the higher affinity of the probe to the target, the probe concentration might be lower.
|LNA primers and probes
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