What Is an siRNA Duplex and How Does It Work?

Small interfering RNA, or siRNA, is a molecule used by cells to regulate gene expression, functioning as a precise tool to selectively “turn off” or silence specific genes. This molecule is a duplex, composed of two strands of RNA bound together. Scientists have learned to harness this cellular mechanism, designing synthetic siRNA molecules to target almost any gene. This capability allows for the detailed study of gene functions and provides a basis for new therapeutic strategies, preventing a particular protein from being made.

The Molecular Structure of an siRNA Duplex

An siRNA duplex has a distinct molecular architecture directly related to its function. These molecules are short, typically consisting of 21 to 23 nucleotide base pairs. The duplex is formed by two separate RNA strands that are complementary to each other, creating a structure that resembles a short zipper.

The two strands are known as the guide strand and the passenger strand. The guide strand, also called the antisense strand, contains the sequence that will recognize the target for silencing. The passenger, or sense, strand is complementary to the guide strand and is essential for creating the double-stranded structure that the cell’s machinery initially recognizes. A feature of the siRNA duplex is the presence of short, two-nucleotide overhangs at the 3′ end of each strand, which are important for the molecule’s function.

The Mechanism of Gene Silencing

Gene silencing by an siRNA duplex is a multi-step pathway that occurs within the cell’s cytoplasm. After an siRNA duplex is introduced into a cell, it is recognized by proteins that form the RNA-Induced Silencing Complex, or RISC. The stability of the duplex’s ends and the identity of the nucleotides at the 5′ end influence how efficiently the molecule is loaded into RISC.

Once the siRNA duplex is loaded, the RISC complex unwinds the two RNA strands. The passenger strand, having served its purpose of creating the duplex, is then cleaved and discarded. This leaves the guide strand bound within the activated RISC, which is now ready to find its target.

The guide strand directs the RISC to a specific messenger RNA (mRNA) molecule. The guide strand’s sequence is perfectly complementary to a sequence on the target mRNA, allowing it to bind with high specificity. A protein within the complex, an endonuclease known as Argonaute-2, then cuts the target mRNA. This cleavage leads to its rapid degradation by the cell, which ultimately prevents it from being translated into a protein and silences the gene.

Natural Functions vs. Synthetic Design

The gene-silencing pathway involving siRNA is an evolutionarily conserved process that serves as a natural defense mechanism in many organisms. Its primary role is to protect against viral infections. Many viruses produce long, double-stranded RNA (dsRNA) as part of their replication cycle. Cells recognize this dsRNA as foreign and use an enzyme called Dicer to chop it into smaller, siRNA-sized duplexes. These fragments are then loaded into the RISC complex, programming it to destroy viral RNAs that match the sequence.

Scientists have taken advantage of this natural cellular pathway. By understanding the structural requirements of siRNA, researchers can design and manufacture synthetic siRNA duplexes in the laboratory. These molecules are engineered with a specific sequence to match any gene of interest. When introduced into cells, these custom-designed siRNAs co-opt the cell’s RISC machinery to silence a targeted gene without altering the cell’s DNA.

Applications in Research and Medicine

The ability to silence specific genes on command makes siRNA a useful tool in biological research. Scientists use synthetic siRNAs to perform “gene knockdown” experiments. By temporarily turning off a single gene in cultured cells or animal models, researchers can observe the resulting changes in cellular behavior or physiology. This loss-of-function approach is a powerful method for determining the role a gene plays in processes like cell division, signaling pathways, or disease development.

Beyond research, siRNA technology has emerged as a promising class of therapeutic drugs. The goal is to silence genes that cause or contribute to disease, which has led to the development of FDA-approved medicines. For example, the drug Patisiran (Onpattro) is an siRNA therapeutic for a rare genetic disorder called hereditary transthyretin-mediated amyloidosis. The drug silences the TTR gene, preventing the production of a toxic, misfolded protein and alleviating the disease’s symptoms. This demonstrates the potential of siRNA to treat conditions by targeting their genetic source.