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Experimental RNAi02:15

Experimental RNAi

RNA interference (RNAi) is a cellular mechanism that inhibits gene expression by suppressing its transcription or activating the RNA degradation process. The mechanism was discovered by Andrew Fire and Craig Mello in 1998 in plants. Today, it is observed in almost all eukaryotes, including protozoa, flies, nematodes, insects, parasites, and mammals. This precise cellular mechanism of gene silencing has been developed into a technique that provides an efficient way to identify and determine the...
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A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA
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Published on: December 2, 2009

Nucleoside optimization for RNAi: a high-throughput platform.

Gabor Butora1, Denise M Kenski, Abby J Cooper

  • 1Department of Process Chemistry, Merck & Co., Rahway, New Jersey 07065, USA. gabor_butora@merck.com

Journal of the American Chemical Society
|September 28, 2011
PubMed
Summary
This summary is machine-generated.

Researchers modified the guide strand (GS) backbone in RNA-induced silencing complexes (RISC) to enhance siRNA stability and therapeutic potential. This systematic approach enables rational design of potent, safe, and effective RNA interference therapeutics.

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11:52

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

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Area of Science:

  • Molecular Biology
  • RNA Interference Therapeutics
  • Drug Discovery

Background:

  • The RNA-induced silencing complex (RISC) utilizes a guide strand (GS) for sequence-specific mRNA cleavage.
  • Modifying the GS backbone offers a strategy to modulate RISC activity and improve siRNA properties.

Purpose of the Study:

  • To systematically evaluate modified nucleosides within the GS of RISC.
  • To establish a platform for rational design of improved small interfering RNAs (siRNAs).

Main Methods:

  • Synthesis of phosphoramidites for all four canonical bases with modifications.
  • Sequential evaluation of modified nucleosides at each position of a 21-nucleotide GS.
  • Utilized inosine as a baseline for comparison of sugar-modified nucleosides.
  • Validated the platform using 2'-O-benzyl modification at specific positions.

Main Results:

  • Demonstrated that positions 5, 8, 15, and 19 of the GS can accommodate bulky modifications like 2'-O-benzyl.
  • Established a high-throughput methodology for assessing modified nucleosides.
  • Defined a new activity baseline using inosine substitutions.

Conclusions:

  • The developed high-throughput methodology facilitates hypothesis-driven design of siRNAs.
  • Enables the creation of potent, immunologically silent, and stable siRNAs for therapeutic use.
  • Advances the development of next-generation RNA interference-based therapies.