Small interfering RNA (siRNA) is a class of small, double-stranded ribonucleic acid molecules, typically 20 to 25 base pairs in length. These molecules are central to a natural biological process called RNA interference (RNAi). The primary function of siRNA is to silence specific genes by intercepting the genetic message, known as messenger RNA (mRNA), after it has been transcribed from DNA. By targeting this intermediate molecule, siRNA prevents the final step of the genetic process: the creation of a functional protein. This precise mechanism offers scientists a highly effective way to turn off genes linked to various diseases.
The Mechanism of Gene Silencing
Gene silencing begins when the double-stranded siRNA molecule is incorporated into the RNA-Induced Silencing Complex (RISC). Once loaded, RISC unwinds the siRNA, retaining one strand designated as the “guide strand” while the other “passenger strand” is degraded. The RISC, armed with the single-stranded guide RNA, then actively scans the cell’s environment for messenger RNA (mRNA) molecules. It specifically seeks mRNA that possesses a perfectly complementary sequence to the guide strand.
The precision of this mechanism relies on exact base-pairing between the siRNA guide strand and the target mRNA. When RISC locates a fully complementary mRNA, the Argonaute protein, a central component of RISC, acts as a molecular scissor. Argonaute cleaves the target mRNA’s phosphodiester backbone, cutting the genetic message into two pieces. Cleaving the mRNA strand marks it for immediate destruction by cellular enzymes.
This destruction ensures the genetic information cannot be read by the ribosome, the structure responsible for protein synthesis. Since the mRNA is degraded before the protein can be manufactured, the expression of the corresponding gene is silenced. The RISC complex remains intact and is recycled to bind and cleave additional copies of the target mRNA, making the silencing process highly efficient.
Natural Roles of siRNA in Cellular Function
The RNA interference pathway is an ancient, naturally occurring mechanism in most eukaryotic organisms, serving as an intrinsic cellular defense system. It primarily protects against invading genetic material, such as viruses. Many viruses produce double-stranded RNA during their replication cycle inside the host cell. Cellular enzymes, such as Dicer, recognize this foreign double-stranded RNA and cleave it into short siRNA fragments. These siRNAs initiate the silencing pathway, targeting and destroying the viral RNA to neutralize the infection before it can spread widely. This mechanism is robust in plants, insects, and worms, and contributes to defense in mammalian cells.
Beyond antiviral immunity, the siRNA pathway maintains the integrity of the host’s genetic code. It controls the activity of mobile genetic elements, known as transposons, which are segments of DNA that can “jump” to different locations. By silencing the genes required for transposon mobility, the cell prevents these elements from causing harmful mutations or genomic instability. This regulatory function is a fundamental aspect of genome surveillance.
Therapeutic Applications of siRNA
The specificity of siRNA’s gene-silencing ability has positioned it as a class of therapeutic agents known as RNAi therapeutics. The concept involves designing a synthetic siRNA molecule complementary to the mRNA of a known disease-causing gene. Introducing this engineered molecule into a patient allows the problematic gene to be “turned off” precisely. This approach offers a way to treat diseases resulting from the overproduction of a harmful protein or the expression of a mutated gene.
Approved siRNA Drugs
Several FDA-approved drugs utilize this technology, demonstrating its clinical viability. Patisiran, one of the first approved siRNA drugs, treats the polyneuropathy of hereditary transthyretin-mediated amyloidosis. It works by silencing the transthyretin gene, reducing the production of the abnormal protein that accumulates in tissues.
Inclisiran is another successful example, targeting the mRNA for the PCSK9 protein to significantly lower LDL cholesterol. This drug has a prolonged effect, often requiring only two doses per year after the initial regimen. The therapeutic potential extends to viral infections like hepatitis B and specific cancers, where silencing a gene that promotes tumor growth or viral replication can halt disease progression.
The Challenge of Cellular Delivery
The clinical success of siRNA has been complicated by significant biological obstacles. The primary hurdle is delivering the fragile RNA molecule safely and effectively into the cytoplasm of target cells. Naked siRNA injected into the bloodstream is rapidly degraded by enzymes called nucleases and quickly cleared by the kidneys. To overcome this, researchers developed sophisticated delivery systems to protect the therapeutic cargo.
Delivery Systems
One successful method involves encapsulating the siRNA within Lipid Nanoparticles (LNPs). These tiny spheres of fat shield the RNA from degradation and help the siRNA enter the cell by fusing with the cell membrane. This mechanism has been particularly effective for targeting the liver.
An alternative strategy is the use of chemical conjugates, such as the N-acetylgalactosamine (GalNAc) conjugate. This method attaches the siRNA molecule to a GalNAc ligand, which specifically binds to receptors on the surface of liver cells. This targeted approach allows for subcutaneous injection and efficient delivery to the liver, making it suitable for treating liver-centric diseases.