RNA interference allows the knockdown of gene expression in cells, for example, in cell cultures. During RNA interference (RNAi), double-stranded RNA (dsRNA) induces sequence-specific gene silencing by targeting mRNA for degradation. For the use of siRNA in cell cultures, cells need to be evaluated for the expression level of the gene of interest. For optimized siRNA knockdown conditions, cell lines expressing relatively high levels of the targeted gene need to be selected.
RNAi is a widespread natural gene-silencing phenomenon conserved across fungi, plants, and animals. Exogenous siRNAs, endogenous small RNAs, microRNAs (miRNAs), and piwi-interacting RNAs induce RNAi. They are involved in several cellular processes, such as cell growth, tissue differentiation, hetero-chromatin formation, and cell proliferation.
RNAi enables the knockdown of gene expression to study the effect of loss-of-function mutations. RNAi has a broad therapeutic potential for various human diseases, including infection and cancer. The use of synthetic siRNAs shows excellent promise in the development of RNA-based therapeutics. However, challenges with delivery and harmful off-target effects will need to be resolved.
Also, RNAi allows the silencing of individual genes post-transcriptionally to study the cellular function of genes. RNAi, when used in cell cultures, allows studying the function of individual genes.
Introducing short interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) into cells allows specific silencing of the expression of a target gene to study the effects of gene knockdowns on cellular processes, such as cell growth, differentiation, and death.
The introduction of double-stranded RNA (dsRNA) into cells triggers RNAi. In the cell, dsRNA is first recognized and processed into 21 to 23 base-pair small interfering RNAs (siRNA) by the RNase III family ribonuclease Dicer. Next, the resulting short interfering RNAs are incorporated into the RNA-inducing silencing complex (RISC), where they direct RISC to the target RNA. The nuclease complex RISC is responsible for destroying the target RNA, resulting in gene silencing in a sequence-specific manner.
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Structure of human Dicer Platform-PAZ-Connector Helix cassette in complex with 14-mer siRNA having 5'-pUU and UU-3' ends (2.55 Angstrom resolution)
https://www.rcsb.org/3d-view/4NH5/1
Tian et al. (2014) solved crystal structures of the human Dicer "platform-PAZ-connector helix" cassette in complex with small interfering RNAs (siRNAs).
The structures have two adjacently positioned pockets: a 2 nt 3'-overhang-binding pocket within the PAZ domain (3' pocket) and a phosphate-binding pocket within the platform domain (phosphate pocket). The structures revealed that the transition from the cleavage-competent to the postulated product release/transfer state may involve the release of the 5'-phosphate from the phosphate pocket while retaining the 3' overhang in the 3' pocket.
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How are siRNAs produced?
Chemical synthesis: The chemical synthesis of siRNAs allows the production of 21 to 22 base-pair siRNA oligonucleotide duplexes. Chemical synthesis is a relatively simple and quick way to generate siRNAs.
In vitro transcription (IVT): IVT uses T7 RNA polymerase to produce siRNAs.
Endogenous expression: The endogenous expression of siRNAs produces short hairpin RNAs (shRNAs)delivered to cells via plasmids, viral or bacterial vectors.
RNAi, as a tool, allows for studying gene function in cell cultures. Introducing siRNAs or short hairpin RNAs (shRNAs) into cells specifically silences the expression of a target gene. RNAi used in cell cultures allows studying the effects of gene knockdown on cellular processes, such as cell growth, differentiation, and death.
How are siRNAs delivered into cells?
The delivery of chemically synthesized siRNAs to mammalian cells is possible through several strategies, such as direct conjugation to cell-surface binding ligands, encapsulation into lipids, and electroporation. However, plasmid-based shRNA vectors are usually delivered with the help of lipids or electroporation, and infection allows the delivery of virus-based vectors into cells.
Three principal delivery methods are possible:
- Delivery of naked siRNA.
- Delivery using siRNA packaged in lipids.
- Delivery as siRNA-conjugates.
During transfection, siRNAs or shRNAs are introduced into cells using chemical transfection, electroporation, or lipofection. However, transduction introduces nucleic acids into cells with the help of a viral vector.
RNAi has several advantages over other gene silencing methods, such as antisense oligonucleotides and gene knockout mice. RNAi is more efficient and specific than antisense oligonucleotides, and it does not require the generation of knockout mice. RNAi is also a relatively quick and easy method to perform.
RNAi has been used to study various biological processes, including cell growth, differentiation, death, and signaling. It has also enabled the development of new therapeutic approaches for multiple diseases, such as cancer and viral infections.
Benefits of using RNAi in cell cultures:
- Specificity: RNAi allows silencing the expression of a single gene without disturbing the expression of other genes.
- Efficiency: RNAi can efficiently silence gene expression, even for genes expressed at low levels.
- Versatility: RNAi allows silencing gene expression in various cell types, including primary cells, immortalized cell lines, and stem cells.
- Speed: RNAi enables silencing gene expression within a few hours or days.
Challenges of using RNAi in cell cultures:
- Off-target effects: siRNAs and shRNAs can sometimes target unintended genes, leading to off-target effects.
- Delivery: Delivery of siRNAs and shRNAs into cells can be challenging, especially for primary and stem cells.
- Stability: Endogenous enzymes in cells can degrade siRNAs and shRNAs, limiting their duration of action.
Despite these challenges, RNAi is a powerful tool for studying gene function in cell cultures. With careful design and optimization, RNAi can generate reliable and reproducible results.
References
Han H. RNA Interference to Knock Down Gene Expression. Methods Mol Biol. 2018;1706:293-302. [PMC]
Mocellin S, Provenzano M. RNA interference: learning gene knock-down from cell physiology. J Transl Med. 2004 Nov 22;2(1):39. [PMC]
Paddison PJ, Caudy AA, Hannon GJ. Stable suppression of gene expression by RNAi in mammalian cells. Proc Natl Acad Sci U S A. 2002 Feb 5;99(3):1443-8. [PMC]
Paddison PJ, Hannon GJ. RNA interference: the new somatic cell genetics? Cancer Cell. 2002 Jul;2(1):17-23. [Cancer-Cell]
Perrimon N, Ni JQ, Perkins L. In vivo RNAi: today and tomorrow. Cold Spring Harb Perspect Biol. 2010 Aug;2(8):a003640. [PMC]
Yang C, Qiu L, Xu Z. Specific gene silencing using RNAi in cell culture. Methods Mol Biol. 2011;793:457-77. [PMC]
Huppi K, Martin SE, Caplen NJ.; Defining and assaying RNAi in mammalian cells. Mol Cell. 2005 Jan 7;17(1):1-10. [Cell[
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