How to design BNA oligonucleotide

BNA3 is “2',4'-BNA^NC (2'-O,4'-aminoethylene bridged nucleic acid)”, which has 2',4'-BNA^NC[NH], 2',4'-BNA^NC[NMe] and 2',4'-BNA^NC[NBn] version. Right now, the BNA synthesized by our company is 2',4'-BNA^NC[NMe] version with the structure below. 2',4'-BNA^NC[NH] version will also be available within a matter of days.

BNA oligonucleotide (BNA3) can be synthesized with  either 100% BNA3 bases or a mixture of BNA/DNA, BNA/RNA bases or other chimeric design.  However, only oligonucleotides that are between 7 and 10 mers in length can be made up of 100% BNA.  If an oligonucleotide is longer, the 100% BNA oligo may bind tightly to itself (self-complement) rather than to the target sequence.  Therefore, for oligonucleotides longer than 15 mers, we recommend decreasing the BNA content as the oligonucleotide increases in length, as follows :
 

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

• BNA 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 BNA bases. BNA hybridizes vary when several consecutive residues are substituted with BNA bases.
• Typical primer length of 18mer should not contain more than eight BNA bases.
• Each BNA base increases the T_m by approximately 2-4 ^oC.
• Do not use blocks of BNA near the 3' end.
• Keep the GC-content between 30-60%.
• Avoid stretches of more than 3 G or C DNA or BNA bases
• T_m of the primer pairs should be nearly equal.

In vivo Knockdown

For in vivo knockdown, consider using  phosphorothioate backbones. Also consider conjugated cholesterol oligos or cell penetrating peptides for better uptake in the cell.

Clamp Oligo

The ratio of BNA modification in the clamp oligos  varies by the type of BNA-NC analogs used, oligo length, GC%, mutation kind, T_m value, etc. Generally, more than 50 or 15 mer DNA oligos should be used and phosphorylation, spacer or dideoxy terminal modifier are used to stop primer extension.

Design Guidelines for SNP Microarray Genotyping

Guidelines for designing BNA 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 BNA 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 BNA 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

• BNA 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 four BNA bases.  BNA hybridized very tightly when several consecutive residues are substituted with BNA bases.

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

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

• Typical primer length of 18mer should not contain more than eight BNA bases.

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

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

• Keep the GC-content between 30-60%

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

• T_m of the primer pairs should be nearly equal.

BNA Design Guidelines for Allele Specific PCR

For improvement of allele specific PCR a single BNA nucleotide should be placed in the terminal 3' or the 3'-1 position.  In both cases, the BNA 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 BNA substituted labeled probes: 15-18 nucleotides (Please note that these are 5-8 bases shorter than the corresponding DNA probes).

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

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

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

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

• Avoid BNA 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.

• Keep the GC-content between 30-60%.

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

BNA (Bridged Nucleic Acids -BNA3) is licensed by Bio-Synthesis, need help with BNA design? Contact us and briefly describe your application and project specification.