Product Sheet

General LNA Oligonucleotide Design Guidelines

1. 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).
2. Avoid stretches of more than 4 LNA bases. LNA hybridizes very tightly when several consecutive residues are substituted with LNA bases.
3. Avoid LNA self-complimentarity and complementarity to other LNA containing oligonucleotides in the assay. LNA binds very tightly to other LNA residues.
4. Typical primer length of 18mer should not contain more than 8 LNA bases.
5. Each LNA bases increases the Tm by approximately 2-4oC.
6. Do not use blocks of LNA near the 3’ end.
7. Keep the GC-content between 30-60 %.
8. Avoid stretches of more than 3 G DNA or LNA bases.
9. Tm of the primer pairs should be nearly equal.

For very specific or novel assay settings, design rules may have to be established empirically, but following the above recommendations will provide a good start.

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.

1. Capture probes should be approximately 12 bp in length.
2. 2-3 LNA bases should be positioned directly at the SNP site.
3. 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.
4. A Tm of approximately 65ºC is recommended.
5. No LNA bases should be positioned in palindrome sequences (GC base pairs are not allowed, while AT base pairs are less critical).

Design Guidelines for PCR primers

1. 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).
2. Avoid stretches of more than 4 LNA bases. LNA hybridizes very tightly when several consecutive residues are substituted with LNA bases.
3. 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 unspecific annealing of the 3’ end).
4. Avoid LNA self-complimentarity and complementarity to other LNA containing oligonucleotides in the assay. LNA binds very tightly to other LNA residues.
5. Typical primer length of 18mer should not contain more than 8 LNA bases.
6. Each LNA bases increases the Tm by approximately 2-4oC.
7. Do not use blocks of LNA near the 3’ end.
8. Keep the GC-content between 30-60 %.
9. Avoid stretches of more than 3 G DNA or LNA bases.
10. Tm 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 given above.

Design guidelines for Real Time QPCR Probes

1. The 3’-end of the dual labeled probe should be blocked with PO4, NH2 or a blocked nucleotide to prevent extension unless a quencher or dye is placed at the 3’ end.
2. Tm of the dual labeled probe should optimally be 10 °C higher that Tm of the forward primer. For single mutation detection the Tm-difference should be 7 °C.
3. Typical Tm of PCR primers for dual labeled assays: 58-60 °C.
4. Typical Tm of dual labeled probes: 65-70 °C (i.e. slightly lower than the extension temperature).
5. Optimal length of LNA substituted dual labeled probes: 15-18 nucleotides (Please note that these are 5-8 bases shorter than the corresponding DNA probes).
6. Maintain Tm with LNA substitutions to match the Tm of the corresponding longer DNA probe.
7. Substitute every third base with LNA in the central segment of the probe. Usually 4-6 LNA substitutions are required to obtain a useful Tm.
8. Avoid stretches of more than 3 G DNA or LNA bases.
9. When detecting single nucleotide mutations, select the probe sequence so that the mutation is located centrally in the probe. Make a single LNA substitution at the position of the single nucleotide mutation.
10. Avoid LNA substitutions participating in formation of secondary structures.
11. Position the dual labeled probe as close as possible to the forward primer.
12. Avoid Guanine (G) in the 5’-position next to the fluorophore.
13. Select the strand giving the lowest concentration of G’s in the probe.
14. Avoid longer stretches of identical nucleotides and especially G’s.
15. Keep the GC-content between 30-60 %.
16. All fluorescent dyes offered by Gene Link 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).

Design guidelines for siRNA containing LNA bases

1. At the 5’ and 3’-end 2-3 bases can be substituted with LNA bases to impart resistance to exonuclease degradation.
2. Substitute every third base with LNA in the central segment of the sense strand. Avoid stretches of more than 3 G RNA or LNA bases.
3. Avoid LNA substitutions participating in formation of secondary structures.
4. Substitute with 2’F U and 2’F C to impart resistance to RNase.
5. Place cholesterol at 3’ end for ease of uptake.

Single strand antisense oligos

1. LNA-DNA-LNA gapmers. Substitue 4 LNA bases at the ends with 8-10 DNA bases in the middle. This activates RNase H activity.
2. ~10 times more stable than siRNA but ~6 fold less knockout
3. Place cholesterol at 3’ end for ease of uptake.
4. The IC values of different modified antisense oligos are given below. A combination of various modifications may lead to higher stability and longer half life.
 

Design Guidelines for FISH Probes

1. LNA substituted probes are excellent improvements in the design of FISH probes.
2. Design 24 to 30mer probes.
3. 5’ Biotin, Cy3, Cy5 or any other fluorescent dye can be used.
4. Substitute with LNA bases at every second or third base.
5. Avoid stretches of more than 3 G or C LNA bases.
6. Avoid LNA self-complimentarity and complementarity to other LNA containing oligonucleotides in the assay. LNA binds very tightly to other LNA residues.
The above guidelines are for all initial FISH probe design. Design rules may have to be established empirically for very specific or novel assay settings, but following the above recommendations will provide a good start.
 

*License Agreement: Locked-nucleic Acid (LNA) phosphoramidites are protected by EP Pat No. 1013661, US Pat No. 6,268,490 and foreign applications and patents owned by Exiqon A/S. Products are made and sold under a license from Exiqon A/S. Products are for research purposes only. Products may not be used for diagnostic, clinical, commercial or other use, including use in humans. There is no implied license for commercial use, including contract research, with respect to the products and a license must be obtained directly from Exiqon A/S for such use.