Small interfering RNA (siRNA) regulates gene expression by utilizing the cell’s natural defense mechanism known as RNA interference (RNAi). This technology employs a synthetic molecule designed to precisely interfere with the genetic instructions responsible for creating a specific protein. By introducing this molecule, scientists can effectively silence a targeted gene, preventing its associated protein from being produced. This method offers a powerful tool for research and a promising pathway for developing therapies.
The Unique Structure of the siRNA Duplex
The small interfering RNA molecule is defined by its double-stranded composition, forming a duplex structure typically 20 to 25 base pairs in length. This architecture is chemically synthesized to mimic an intermediate structure naturally recognized by the cellular machinery responsible for gene silencing. The duplex includes short, unpaired extensions, known as two-nucleotide 3′ overhangs, which facilitate recognition and processing within the cell.
The duplex is composed of two distinct strands: the passenger strand (sense strand) and the guide strand (antisense strand). The passenger strand is temporary and ultimately discarded by the cell’s processing machinery. The guide strand is the working component, as its sequence is complementary to the target messenger RNA (mRNA) intended for destruction. The selection of the active guide strand is often determined by the relative thermodynamic stability at the ends of the duplex.
How siRNA Silences Target Genes
Gene silencing occurs through the RNA interference (RNAi) pathway. Once the siRNA duplex enters the cytoplasm, it is incorporated into the RNA-Induced Silencing Complex (RISC). Within the RISC, the siRNA duplex undergoes unwinding and separation of its two strands.
The passenger strand is cleaved and released from the complex, while the guide strand remains bound to the Argonaute 2 (Ago2) protein, the core component of RISC. This single guide strand directs the RISC to the specific messenger RNA (mRNA) molecule that shares a perfectly complementary sequence. This precise base-pairing provides siRNA with high specificity, allowing it to target one gene without affecting others.
Upon locating the target mRNA, the Ago2 protein within the RISC acts as an endonuclease, cleaving the phosphodiester backbone of the mRNA at the site of complementarity. This cleavage renders the mRNA unusable and signals for its degradation by cellular enzymes. The destruction of the mRNA transcript prevents the translation of the genetic code into protein, achieving gene silencing. The RISC complex, still bound to the guide strand, is then recycled to find and cleave additional copies of the target mRNA, which amplifies the silencing effect.
Current Medical Applications
The ability to silence disease-causing genes makes siRNA a powerful therapeutic tool with expanding applications. One of the earliest successes was the treatment of hereditary transthyretin-mediated (hATTR) amyloidosis, a rare genetic disorder where an unstable protein accumulates. An approved siRNA therapy for this condition works by silencing the gene responsible for the faulty transthyretin protein, reducing its production.
Another application is in cardiovascular health, specifically for managing high cholesterol. An FDA-approved siRNA therapy targets the messenger RNA for PCSK9, a protein that regulates cholesterol levels. Silencing the PCSK9 gene significantly lowers harmful LDL cholesterol in patients at increased risk of heart disease.
Researchers are also exploring siRNAs for viral infections, such as hepatitis B, and for oncology. In cancer treatment, siRNAs can be designed to silence oncogenes that drive the growth and spread of cancer cells.
Getting siRNA into the Cell
A primary challenge for using siRNA as a drug is delivery, as the molecule is large, hydrophilic, and carries a negative electrical charge. These characteristics prevent naked siRNA from easily crossing the cell membrane to reach the cytoplasm where the RISC complex resides. Furthermore, unprotected siRNA is rapidly degraded by nucleases, enzymes that destroy foreign nucleic acids in the bloodstream.
To overcome these hurdles, the most successful strategy involves encapsulating the siRNA within a protective shell, most commonly a Lipid Nanoparticle (LNP). LNPs are composed of various lipids, including an ionizable cationic lipid that is neutrally charged at physiological pH. This lipid becomes positively charged in the acidic environment of the endosome, facilitating the release of the siRNA payload into the cytoplasm. The LNP platform is effective for targeting the liver, as this organ naturally processes lipid-based particles.