What is Short Hairpin RNA (shRNA) and How It Works?

What is shRNA?

Short Hairpin RNA (shRNA) is a specific type of hairpin RNA that is widely used in gene silencing techniques through the RNAi mechanism. shRNA is specifically made to degrade certain mRNA molecules and thus repress or mute the gene expression of that specific gene. The technology has in recent years been established as a powerful tool for functional genomics, therapeutic discovery and molecular biology research. It has a hairpin structure with double stranded stem and loop. shRNA is a powerful tool in molecular biology, enabling researchers to silence specific genes and study their functions in detail. In both basic research and the discovery of new therapeutic approaches, its capability to offer a stable effective gene knockdown is an invaluable tool.

Common shRNA expression cassettes driven by a Pol II promoter or a Pol III promoter, and the subsequent processing pathway. (Sheng, P.K.; et al, 2020)

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Hairpin RNA

Hairpin RNA (hpRNA)  - a molecule that unfolds back on itself and forms the shape of an open hairclip, with double-stranded DNA at its stem and loop. The most common secondary structure is depicted here, it looks somewhat like a hairpin or pin with loop at the top. Hairpin RNAs are important in several biological processes specifically, the gene regulation through RNAi pathways. Hairpin RNA serves several important functions in cellular biology, primarily through its involvement in gene silencing and regulation. Hairpin RNA is a versatile and powerful tool in molecular biology, essential for regulating gene expression and advancing our understanding of genetic functions and interactions. Its unique structure and ability to induce RNA interference make it invaluable in research and therapeutic applications.

What is RNA Hairpin Structure?

The RNA hairpin structure consists of a double-stranded stem formed by base pairing between complementary sequences within the RNA strand, and a single-stranded loop that connects the two ends of the stem. The stability of the hairpin structure is influenced by the length and sequence of the stem and loop regions. This structure is crucial for the function of many non-coding RNAs, including microRNAs (miRNAs) and shRNAs, as it facilitates the recognition and processing by cellular machinery.

• RNA Hairpin Stem

The stem is formed by base pairing between complementary sequences within a single RNA molecule. This pairing results in a stable double-stranded region, typically composed of 19 to 29 base pairs (bp) in length. The stability of the stem is crucial for maintaining the overall structure of the hairpin.

• RNA Hairpin Loop

The loop connects the two ends of the stem and is characterized by a single-stranded region where the RNA backbone folds back on itself. The loop is generally short, containing anywhere from 4 to 10 nucleotides, though longer loops can also exist. This single-stranded region provides flexibility to the hairpin structure. The loop can vary in length and sequence, and its flexibility and conformation are important for the biological function of the hairpin RNA. In the context of shRNA, the loop is essential for proper folding and processing of the RNA molecule into its active form.

What is the Function of shRNA?

The primary function of shRNA is to mediate gene silencing through the RNAi pathway. By designing shRNAs to target specific mRNA sequences, researchers can selectively reduce the expression of genes of interest. This technology is particularly useful for studying gene function, validating drug targets, and developing RNA-based therapeutics.

shRNA-Mediated Gene Silencing

• Hairpin RNA-mediated gene silencing leverages the natural RNAi pathway to downregulate gene expression. This method has been employed to study the function of various genes in different biological contexts.  shRNA functions by harnessing the natural RNAi pathway within cells to specifically silence target genes. The process typically involves several key steps:
• Design and Synthesis: shRNAs are synthetic RNA molecules engineered to mimic the structure of endogenous microRNA (miRNA) precursors. They consist of a stem-loop structure where one arm of the stem is complementary to the target mRNA sequence.
• Cellular Uptake: Once introduced into cells via transfection or viral vectors, shRNAs are processed by cellular machinery.
• Dicer Processing: The enzyme Dicer recognizes and cleaves the shRNA at the loop region, generating a short double-stranded RNA duplex.
• Loading into RISC: One strand of the resulting duplex, known as the guide strand, is loaded into the RNA-induced silencing complex (RISC).
• Target mRNA Binding: The guide strand within the RISC complex then guides the RISC to complementary sequences on the target mRNA.
• Gene Silencing: Binding of the RISC to the target mRNA can lead to mRNA degradation or translational repression, thereby silencing gene expression.

shRNA vs siRNA

Short hairpin RNA (shRNA) is a type of RNA that forms a tight hairpin turn structure. It is typically encoded by a plasmid or viral vector and expressed within the cell's nucleus. Once transcribed, the shRNA is processed into a mature double-stranded RNA by the enzyme Dicer, which then enters the RNA-Induced Silencing Complex (RISC) to guide the degradation of target mRNA. While small interfering RNA (siRNA) is a double-stranded RNA molecule, typically 20-25 nucleotides long. It is usually synthesized chemically and introduced directly into the cytoplasm of cells. siRNA guides the RISC complex to the target mRNA, leading to its cleavage and subsequent degradation.

Applications of shRNA

Short hairpin RNA (shRNA) is a versatile tool with diverse applications in molecular biology, biotechnology, and therapeutic development. Here's the key applications of shRNA:

Functional Genomics and Gene Discovery

shRNA enables selective gene silencing, helping researchers study gene function and establish causal relationships in disease mechanisms and developmental pathways.

Disease Modeling and Pathway Analysis

By targeting disease-associated genes, shRNAs aid in creating disease models to study molecular mechanisms and screen potential therapies.

Therapeutic Development and Gene Therapy

shRNA holds promise for treating genetic disorders, viral infections, and cancer by silencing disease-causing genes or viral genomes.

Biotechnological and Agricultural Applications

In biopharmaceuticals, shRNAs enhance protein production, while in agriculture, they improve crop traits and resistances.

Basic Research and Tool Development

shRNA is essential in basic research, allowing precise gene manipulation and validation of drug targets.

Reference

1. Sheng, P.K.; et al. Short Hairpin RNAs for Strand-Specific Small Interfering RNA Production. Front. Bioeng. Biotechnol. 2020, 8.

What is Short Hairpin RNA (shRNA) and How It Works?

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