Terminal Duplex Stability and Nucleotide Identity Differentially Control siRNA Loading and Activity in RNA Interference

Phillip A. Angart, Rebecca J. Carlson, Kwasi Adu-Berchie, S. Patrick Walton

    Research output: Contribution to journalArticle

    Abstract

    Efficient short interfering RNA (siRNA)-mediated gene silencing requires selection of a sequence that is complementary to the intended target and possesses sequence and structural features that encourage favorable functional interactions with the RNA interference (RNAi) pathway proteins. In this study, we investigated how terminal sequence and structural characteristics of siRNAs contribute to siRNA strand loading and silencing activity and how these characteristics ultimately result in a functionally asymmetric duplex in cultured HeLa cells. Our results reiterate that the most important characteristic in determining siRNA activity is the 5′ terminal nucleotide identity. Our findings further suggest that siRNA loading is controlled principally by the hybridization stability of the 5′ terminus (Nucleotides: 1-2) of each siRNA strand, independent of the opposing terminus. Postloading, RNA-induced silencing complex (RISC)-specific activity was found to be improved by lower hybridization stability in the 5′ terminus (Nucleotides: 3-4) of the loaded siRNA strand and greater hybridization stability toward the 3′ terminus (Nucleotides: 17-18). Concomitantly, specific recognition of the 5′ terminal nucleotide sequence by human Argonaute 2 (Ago2) improves RISC half-life. These findings indicate that careful selection of siRNA sequences can maximize both the loading and the specific activity of the intended guide strand.

    Original languageEnglish (US)
    Pages (from-to)309-317
    Number of pages9
    JournalNucleic Acid Therapeutics
    Volume26
    Issue number5
    DOIs
    StatePublished - Oct 1 2016

    Profile

    RNA Interference
    Small Interfering RNA
    Nucleotides
    Bibliography of Medicine
    RNA-Induced Silencing Complex
    Gene Silencing
    HeLa Cells
    Half-Life
    Cultured Cells
    Proteins
    Cyclic AMP Receptor Protein
    Blood Flow Velocity

    Keywords

    • Ago2
    • duplex thermodynamics
    • functional asymmetry
    • RNAi
    • siRNA
    • terminal nucleotide

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Medicine
    • Molecular Biology
    • Genetics
    • Drug Discovery

    Cite this

    Terminal Duplex Stability and Nucleotide Identity Differentially Control siRNA Loading and Activity in RNA Interference. / Angart, Phillip A.; Carlson, Rebecca J.; Adu-Berchie, Kwasi; Walton, S. Patrick.

    In: Nucleic Acid Therapeutics, Vol. 26, No. 5, 01.10.2016, p. 309-317.

    Research output: Contribution to journalArticle

    Angart, Phillip A.; Carlson, Rebecca J.; Adu-Berchie, Kwasi; Walton, S. Patrick / Terminal Duplex Stability and Nucleotide Identity Differentially Control siRNA Loading and Activity in RNA Interference.

    In: Nucleic Acid Therapeutics, Vol. 26, No. 5, 01.10.2016, p. 309-317.

    Research output: Contribution to journalArticle

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