LNA Oligonucleotide Lock Nucleic Acid (LNA) LNA Hybridizes

General LNA Oligonucleotide Design Guidelines

LNA Oligos are composed of either 100% Lock Nucleic Acid (LNA) bases or a mixture of LNA and DNA bases.  However, only oliognulceotides that are between 7 and 15 mers in length can be made up of 100% LNA.  If an oligonucleotide is longer, the 100% LNA oligo may bind to itself (self complement) rather than to the target sequence.  Therefore, for oligonucleotides longer than 15 mers, we recommend decreasing the LNA content as the oligonucleotide increases in length, as follows:

If longer sequence is absolutely necessary, we can synthesize client's existing design as chimera with mixture of LNA bases and DNA bases and phosphorothioate modifications only the DNA gap, leaving LNA flanks phosphodiester LNA base linkage. This is the only way we can synthesize longer sequences.

• LNA's should be introduced at the positions where specificity and discrimination is needed (e.g. 3' end in allele specific PCR and in the SNP position in allele specific hybridization probes).
• Avoid stretches of more than 4 LNA bases. LNA hybridizes very tightly when several consecutive residues are substituted with LNA bases.
• Typical primer length of 18mer should not contain more than 8 LNA bases
• Each LNA bases increases the T_m by approximately 2-4 ^oC.
• Do not use blocks of LNA near the 3' end.
• Keep the GC-content between 30-60%
• Avoid stretches of more than 3 Gs or Cs DNA or LNA bases
• T_m of the primer pairs should be nearly equal.

In vivo Knockdown

For in vivo knockdown, consider using complete phosphorothioate backbones, and also consider conjugated cholesterol oligos or cell penetrating peptide for better uptakes in the cell.

Design Guidelines for SNP Microarray Genotyping

Guidelines for designing LNA containing oligonucleotides for genotyping using SNP chip microarrays are listed below.  Please note that all the general design guidelines also apply.  the guidelines should be considered as a rule of thumb.

• Capture probes should be approximately 12 bp in length.

• 2-3 LNA bases should be positioned directly at the SNP site.

• The position of the mismatch in the capture probe is flexible - however, positioning the SNP at the very 3' or 5' end or 1 position from the ends may compromise discrimination.

• A T_m of approximately 65 ^oC is recommended.

• No LNA bases should be positioned in aplindrome sequences (GC base pairs are not allowed, while AT base pairs are less critical).

Design Guidelines for PCR Primers

• LNA's should be introduced at the positions where specificity and discrimination is needed (e.g. 3' end in allele specific PCR and in the SNP position in allele specific hybridization probes).

• Avoid stretches of more than 4 LNA bases.  LNA hybridized very tightly when several consecutive residues are substituted with LNA bases.

• Start by spiking LNA in the 5' end of the primer (allow the 5' end of the primer to anneal at high TM avoiding random priming by un- specific annealing of the 3' end).

• Avoid LNA self-complimentarity and complementarity to other LNA containing oligonucleotides in the assay.  LNA binds very tightly to other LNA residues.

• Typical primer length of 18mer should not contain more than 8 LNA bases.

• Each LNA bases increases the T_m by approximately 2-4 ^oC.

• Do not use blocks of LNA near the 3' end.

• Keep the GC-content between 30-60%

• Avoid stretches of more than 3 G DNA or LNA bases.

• T_m of the primer pairs should be nearly equal.

Design guidelines for Allele Specific PCR

For improvement of allele specific PCR a single LNA nucleotide should be placed in the terminal 3' or the 3'-1 position.  In both cases the LNA base should correspond to the position of the polymorphism. Follow general design guidelines above.

Design guidelines for Real Time qPCR Probes

• The 3'-end of the labeled probe should be blocked with PO4, NH2 or a blocked nucleotide to prevent extension.

• T_m of the labeled probe should optimally be 10 ^oC higher that T_m of the forward primer. For single mutation detection the T_m-difference should be 7 ^oC

• Typical T_m of  PCR primers for labeled assays: 58-60 ^oC

• Typical T_m of labeled probes: 65-70 ^oC (i.e. slightly lower than the extension temperature).

• Optimal length of LNA substituted labeled probes: 15-18 nucleotides (Please note that these are 5-8 bases shorter than the corresponding DNA probes).

• Maintain T_m with LNA substitutions to match the T_m of the corresponding longer DNA probes.

• Substitute every third base with LNA in the central segment of the probe.  Usually 4-6 LNA substitutions are required to obtain a useful T_m.

• Avoid stretches of more than 3 G DNA or LNA bases.

• When detecting single nucleotide mutations, select the probe sequence so thtat the mutation is located centrally in the probe.  Make a single LNA substitution at the position of the single nucleotide mutation.

• Avoid LNA substitutions participating in formation of secondary structures.

• Position the labeled probe as close as possible to the forward primer.

• Avoid Guanine (G) in the 5'-position next to the fluorophore.

• Select the strand giving the lowest concentration of G's in the probe.

• Avoid longer stretches of identical nucleotides and especially G's.

• Keep the GC-content between 30-60%

• All fluorescent dyes offered by Bio-Synthesis can be conjugated to LNA containing probes. (FAM, TET, HEX, TAMRA, ROX, CY3, CY3.5, Texas Red, Cy5, Cy5.5, Cy7 and Alexa series dyes and more...)

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