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Month: January 2016

Intronic off-target effects with antisense oligos

Intronic off-target effects with antisense oligos

Undecided on whether to use silencing RNAs (siRNAs) or antisense oligos (ASOs) for your RNA interference experiments? Read on.

ASOs are single-stranded DNA oligonucleotides which downregulate specific RNA by hybridizing with it, forming a heteroduplex recognizable by RNase H1. RNase H1 is found in the nucleus, hence ASOs are often used to target nuclear-localized RNAs such as non-coding RNA.

A recent paper however highlighted that since RNase H1 is found in the nucleus, intronic sequences may also fall prey to ASO-mediated silencing, exposing ASOs to a much larger array of off-target sequences. Indeed, a quantitative PCR analysis of chemically modified ASOs (marketed as GapmeRs) targeting a gene called BACH1 showed a surprisingly high number of positive off-target effects even with transcripts of low sequence identity (PHF6 – 4 nt gap with exon; 3 mismatches with intron).

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The dose and time-dependent nature of ASO-mediated RNA downregulation (measured by qPCR) in off-target genes of two ASOs targeting BACH1 is seen here in various cell lines.

ASO-mediated off-target effects were detected against both exonic and intronic regions, though intronic regions proved more susceptible. This is illustrated below where greater off-target activity was seen in chart C (intron-based OTEs) as opposed to chart B (exon-based OTEs).

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Graphical summary of 26 OTEs predicted and tested for GSK2910546A (ASO). Total (A), exonic (B) and intronic (C) off-target effects are grouped by their potency relative to EC50 of intended target (BACH1, 0.3 μM).

Nuclear-localized RNAs are thought to be less susceptible to siRNA-mediated degradation due to a lower expression level of RNA-induced silencing machinery (RISC) components in the nucleus. That is not to say they are not thereSilencing of nuclear-localized RNAs such as 7SK by siRNA also occurs efficiently, indicating RISC-mediated RNAi can take place in the nucleus.

siPOOLs address hybridization-dependent toxicities and have been shown to work with nuclear-localized RNAs. Contact us to try out siPOOLs today.

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References:

Kamola, P. J., Kitson, J. D. A., Turner, G., Maratou, K., Eriksson, S., Panjwani, A., … Parry, J. D. (n.d.). In silico and in vitro evaluation of exonic and intronic off-target effects form a critical element of therapeutic ASO gapmer optimization. Nucleic Acids Research , 43 (18 ), 8638–8650. Retrieved from http://nar.oxfordjournals.org/content/43/18/8638.abstract

Gagnon, K. T., Li, L., Chu, Y., Janowski, B. A., & Corey, D. R. (2016). RNAi Factors Are Present and Active in Human Cell Nuclei. Cell Reports, 6(1), 211–221. doi:10.1016/j.celrep.2013.12.013

Robb, G. B., Brown, K. M., Khurana, J., & Rana, T. M. (2005). Specific and potent RNAi in the nucleus of human cells. Nat Struct Mol Biol, 12(2), 133–137. Retrieved from http://dx.doi.org/10.1038/nsmb886

 

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Simplicity is the ultimate sophistication

Simplicity is the ultimate sophistication

The beauty of the siPOOL strategy is its simplicity.

In this presentation from the  (relatively) early days of Apple, Steve Jobs says that his company’s goal is to serve the one-on-one relationship between a user and his/her computer.

 

Similarly, siPOOLs, are designed to serve the one-on-one relationship between a scientist and his/her RNAi results.

By providing an interpretable result without the need for extensive follow-up work and off-target corrections, siPOOLs make it possible for a scientist to use a single gene list to gain insight into biological function.

We believe, as stated in the brochure to market the Apple II,  that  Simplicity is the ultimate sophistication.

(Note: this quote is popularly, though apparently falsely, attributed to Leonardo da Vinci) .

 

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