RNase H-Active Antisense Oligonucleotide Services
Gapmer antisense chimeras are designed to have both 2'OMe RNA and BNA in the sequence that retain an RNase H activating domain of DNA (or phosphorothioated DNA). As BNA bases confer significant nuclease resistance, we suggest the placement of phosphorothioate modification of only at the DNA gap and leaving the BNA flank as phosphodiester linkages in chimeric BNA Gapmer antisense oligonucleotides. The use of 3'-phosphate is preferred when a BNA base is at the 3' end.
It can also be beneficial to substitute 5-Methyl-dC for dC monomers in the context of CpG motifs. Substitution of 5-methyl dC for dC will slightly increase the Tm of the antisense oligo. Use of 5-Methyl dC in CpG motifs can also reduce the chance of adverse immune responses in vivo.
Bio-Synthesis recommends that all antisense oligos receive RNase free HPLC purification and that oligos undergo a Na+ salt exchange before use in cells or live animals to ensure that salts used in purification are removed.
We offer wide variety of base analog modifications, contact us for items not listed below.
Sample Processing Options:
- In-vitro Basic: budget friendly option allowing many designs to go through the initial screening process.
- In-vitro Standard: RNase free HPLC or PAGE purified oligos suitable for most cell assays. Also available with custom 5’ or 3’ fluorescent labels.
- In-vivo-Ready Gold: BNA gapmer has been processed by counter-ion (Na+) exchange, desalting, sterile filtration, and endo toxin testing.
- In-vivo-Ready Platinum: High quality oligos recommended for experiments with the ultimate goal of in vivo testing or cell lines. The BNA gampers have undergo both in vivo processing as well as dual RNase free HPLC purification.
Additional Services:
Custom Preparative and Analytical Services also available upon request.
Bio-Synthesis is the exclusive provider of synthetic BNAs. The purchase of BNA is for research use only. Bio-Synthesis carries a research use only license, clinical/therapeutic and/or commercial application of BNAs require a separate commercial license. We would be glad to discuss licensing terms with interested parties.
Highest Knockdown, Lowest Off-target Gene Silencing by RNase H-Activate Antisense Oligonucleotide
Artificial regulation of gene expression is an important technique to analyze unknown
biological functions of target genes. In antisense technology, a single-stranded
oligonucleotide added from outside may bind with target mRNA to form oligonucleotide-RNA
duplex. RNase H is the enzyme found in the cytoplasm and nucleus of cells. It hydrolyzes
the RNA of the RNA-DNA heteroduplexes formed after sequence specific binding of
ASOs to their target mRNA (or lncRNA). Once cleaved by RNase H, the degraded
mRNA is not translated into the protein, resulting in down-regulation of the targeted
protein.
However, serious difficulties, such as poor binding ability of added oligonucleotides
with target mRNA or target duplex DNA, and low stability of added oligonucleotides
against nuclease degradation often limit practical applications of the antisense
or antigene technologies in vivo. Other limiting factors such as the occurrence of
toxic side-effects together with non-specific binding causing off-target effects
has stimulate the design of new artificial nucleic acids for the development of
modified oligonucleotides that provide efficient and specific antisense activity
in vivo without exhibiting toxic side-effects.
The use of BNA gapmer chimeric oligonucleotides have advantages of: