Small interfering RNA (siRNA) knockdown is a powerful biological tool used to selectively reduce the expression of specific genes within cells. This process, often referred to as gene silencing, works by interfering with the messenger RNA (mRNA) that carries genetic instructions from DNA to the protein-making machinery of the cell. By targeting and degrading these mRNA molecules, siRNA prevents the production of the corresponding proteins, effectively “turning off” a gene. This temporary and often incomplete reduction in gene expression allows scientists to investigate the functions of individual genes and explore their roles in various biological processes and diseases.
How Genes Are Silenced by siRNA
The mechanism by which siRNA silences genes is rooted in a natural cellular process called RNA interference (RNAi). This pathway begins when double-stranded RNA (dsRNA) enters a cell, either naturally or through experimental introduction. Cellular enzymes, particularly an RNase III-like enzyme called Dicer, recognize and cleave this longer dsRNA into smaller fragments, typically 20-25 nucleotides in length, known as small interfering RNAs (siRNAs).
Once formed, the siRNA molecules are then incorporated into a multi-protein complex called the RNA-induced silencing complex (RISC). Within the RISC, the double-stranded siRNA is unwound, and one of its strands, known as the “guide strand” or antisense strand, is retained, while the other “passenger strand” is degraded. The guide strand directs the RISC to its specific target.
The RISC, now loaded with the guide strand, scans for messenger RNA (mRNA) molecules that have a complementary sequence to the siRNA guide strand. When a perfect or near-perfect match is found, the RISC binds to the target mRNA. The Argonaute 2 (AGO2) protein, a component of the RISC, then cleaves and degrades the target mRNA. This degradation prevents the mRNA from being translated into a protein, leading to a reduction or elimination of the gene’s expression.
Utilizing siRNA in Research and Medicine
siRNA knockdown has become a widely used technique in scientific research to understand gene function. By selectively silencing a gene, researchers can observe the resulting changes in cellular behavior, such as cell growth, response to drugs, or DNA repair mechanisms. This allows for the study of specific gene products and their roles in various biological pathways and disease development.
Beyond basic research, siRNA holds promise for therapeutic applications, particularly in developing treatments for diseases caused by aberrant gene expression. This technology is being explored for viral infections, certain cancers, and genetic disorders. For instance, siRNAs have been investigated to target viral proteins in infections like Ebola, and some siRNA-based drugs have already received regulatory approval.
Patisiran, approved in 2018, is an example of an FDA-approved siRNA-based drug used to treat hereditary amyloidogenic transthyretin (hATTR) amyloidosis with polyneuropathy. Other approved siRNA drugs, such as givosiran, inclisiran, and lumasiran, address conditions like acute hepatic porphyria, high cholesterol, and primary hyperoxaluria type 1, respectively. These advancements highlight the potential of siRNA therapeutics to address previously challenging diseases by precisely targeting and silencing disease-causing genes.
Getting siRNA Inside Cells
A challenge in utilizing siRNA for research and therapeutic applications is effectively delivering these molecules into target cells. As siRNA is a relatively large and negatively charged molecule, it does not easily cross the cell membrane on its own and is susceptible to degradation by enzymes in the bloodstream. Various strategies have been developed to overcome these barriers, broadly categorized into viral and non-viral methods.
Viral vectors, such as adeno-associated viruses (AAVs), are efficient at introducing genetic material into cells. While effective, concerns about immune responses and potential safety issues associated with viral delivery need careful consideration. Non-viral methods offer alternatives, including physical approaches like electroporation, which uses electric fields to temporarily create pores in cell membranes, and ultrasonic techniques that use sound energy to enhance uptake.
Chemical methods are also widely employed, with lipid nanoparticles (LNPs) being a well-established and efficient delivery system. LNPs encapsulate siRNAs, protecting them from degradation and facilitating their entry into cells through processes like endocytosis. Other chemical approaches involve cationic polymers, which bind to siRNA to form nanoparticles, and N-acetylgalactosamine (GalNAc) conjugates, which are particularly effective for liver-targeted delivery due to their recognition by specific receptors on liver cells.
Ensuring Precise Gene Silencing
Achieving precise gene silencing is important for both research accuracy and therapeutic safety. A concern with siRNA is the occurrence of “off-target effects,” where the siRNA inadvertently silences unintended genes. These off-target effects can arise from partial complementarity between the siRNA guide strand and other mRNA sequences.
Such unintended silencing can lead to misleading experimental results in research and potentially adverse side effects in therapeutic applications. To minimize these effects, several strategies have been developed. One approach involves careful siRNA sequence design, focusing on the “seed region” of the guide strand, which influences off-target binding.
Chemical modifications to the siRNA molecule, particularly within the seed region, can weaken unintended interactions while maintaining strong binding to the intended target. Another strategy is the use of siRNA pools, which combine multiple siRNA sequences targeting the same gene; this dilutes the impact of any single off-target effect.