This characteristic can increase oligo circulation time and improve cellular internalization when a delivery tool is not employed. Including extensive PS modification can also promote the antisense oligo to non-specifically bind to proteins.
Microsynth oligo as1 full#
Antisense oligos can include a full PS backbone however, the Tm decreases with each PS bond added. Phosphorothioate (PS) bonds are added to antisense oligonucleotides to protect them from nuclease degradation. Salt exchange followed by end-user ethanol precipitation of the antisense oligo is recommended to mitigate toxicity from residual chemicals that may carry over during synthesis. In these instances, HPLC purification combined with Na + For use in live animals, higher purity oligos may be required. We recommend standard desalt purification for most antisense applications. Immune response to Toll-like receptor 9 (TLR9) in vivo. The use of 5-methyl dC in CpG motifs can also reduce the chance of adverse Substitution of 5-methyl dC for dC will slightly increase the T m of the antisense oligo. It can be beneficial to substitute 5-methyl-dC for dC in the context of CpG motifs.
Microsynth oligo as1 plus#
As manyĢ′- O-modified RNA (such as 2′OMe RNAs and Affinity Plus locked nucleic acid bases) are sensitive to exonuclease degradation, we recommend phosphorothioate modification of the ASO sequence to provide stability (See the "Phosphorothioates The preferred antisense strategy is a "gapmer" design which incorporates 2′- O-modified RNA or Affinity Plus locked nucleic acid bases in chimeric antisense oligos that retain an RNase H activating domain. The use of modified RNA, such as 2′-O-methoxy-ethyl (2′-MOE) RNA, 2′-O-methyl (2′OMe) RNA, or Affinity Plus locked nucleic acid bases in chimeric antisense designs, increases both nuclease stability and affinity (T m) of the antisense oligo to the target RNA. State-of-the-art antisense design employs chimeras with both DNA and modified RNA bases. Phosphorothioate linkages also promote binding to serum proteins, which increases the bioavailability of the ASO while facilitating productive cellular uptake.Ĭhemically modified RNA and DNA bases for chimeric antisense designs We recommend phosphorothioate modification of ASO sequences to provide stability. PS oligos can show greater non-specific protein binding than unmodified phosphodiester (PO) oligos, whichĬan cause toxicity or other artifacts when present at high concentrations. In the IDT ordering system, an asterisk indicates the presence of a phosphorothioate internucleoside linkage. In phosphorothioates, a sulfur atom replaces a non-bridging oxygen The simplest and most widely used nuclease-resistant chemistry available for antisense applications is the phosphorothioate (PS) modification. Many 2′- O-modified RNA (such as 2′OMe RNAs and Affinity Plus locked nucleic acid bases) are sensitive While unmodified oligodeoxynucleotides can display some antisense activity, they are subject to rapid degradation by endo- and exo-nucleases. These ASOs are designed with 2' modifications on the sugar moiety throughout the length of the sequence in order to prevent RNase H activation.
Unlike above, these ASOs create a double-stranded region that sterically blocks splicing factors from binding. Sequence, and therefore, are complementary to the exon and intron junction. Alternative splicing is one of many ways gene expression is modulated to respond to changing environmental conditions or developmental cues. The resulting decrease in RNA levels can be measured using RT-qPCR or RNA-seq.ĪSOs can also be designed to investigate the role of alternatively spliced mRNA. To inhibit gene expression, they are introduced into the cell or organism, where theyīind the target RNA to form an RNA/DNA heteroduplex, which is a substrate for endogenous cellular RNase H (Figure 1). They are designed in antisense orientation to the RNA of interest, hence the name. Long, containing a phosphorothioate-modified DNA segment of at least six bases. ASOs are increasingly used to confirm phenotypes obtained from RNAi-mediated gene silencing experiments, also.ĪSOs are nucleic acid sequences made as synthetic oligonucleotides, usually 15–22–bases Research, genomics, target validation, and drug discovery. Improvements in the design and chemistry of antisense compounds have enabled this technology to become a routine tool in basic
PCR Allele Competitive Extension (PACE) genotyping.Drug target identification via CRISPR screening.
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