What is siRNA and siRNA-seq

What is siRNA?

Small interfering RNA (siRNA) are short, non-coding RNA molecules, typically 20-30 nucleotides long, that play a crucial role in the post-transcriptional regulation of gene expression. siRNA was first discovered in plants and later identified in other organisms, including the model organism Caenorhabditis elegans and mammalian cells. Its discovery was pivotal, changing our understanding of gene regulation and setting the stage for new therapeutic approaches targeting specific genes.

Mechanism of siRNA Function

Processing of siRNA by Dicer

Dicer Structure and Function: The journey of siRNA begins with its processing from long double-stranded RNA (dsRNA) into shorter siRNA molecules by the enzyme Dicer. Dicer is a member of the RNase III family and has distinct domains that include a helicase domain, a PAZ (Piwi/Argonaute/Zwille) domain, two RNase III domains, and a dsRNA-binding domain. These domains collectively facilitate the accurate cleavage of dsRNA into siRNA, a critical first step in the RNAi pathway.

siRNA Formation: After Dicer cleaves the dsRNA, the resulting siRNA molecules are approximately 21-23 nucleotides in length and contain two-nucleotide overhangs at the 3' ends. The siRNA duplex formed consists of a guide strand and a passenger strand, with the guide strand being essential for directing the silencing complex to the target mRNA.

RISC Loading and Activation

RISC Assembly: The siRNA duplex produced by Dicer is subsequently incorporated into the RNA-induced silencing complex (RISC). RISC is a multi-protein complex, with Argonaute (AGO) proteins, particularly AGO2 in humans, serving as the central components. The RISC loading complex (RLC) facilitates the transfer of the siRNA to AGO2.

Strand Selection within RISC: Within RISC, the siRNA duplex undergoes unwinding, leading to the degradation of the passenger strand. The guide strand, selected according to the relative thermodynamic stability of its ends, remains associated with AGO2. Typically, the strand with the less stable 5' end is incorporated as the guide strand, ensuring accurate targeting of the complementary mRNA.

Target Recognition by the Guide Strand

Base Pairing Specificity: The guide strand's role is to navigate the RISC to the target mRNA through a sequence-specific interaction. The guide strand pairs with the complementary sequence on the mRNA, which guarantees that only the intended mRNA is targeted for degradation. This high degree of specificity makes siRNAs highly effective in silencing specific genes.

Mechanics of mRNA Targeting: The precision of siRNA targeting is pivotal to its efficacy. The perfect or near-perfect base pairing required between the guide strand and the target mRNA ensures accurate and efficient gene silencing, minimizing off-target effects. This specific binding is crucial for subsequent mRNA cleavage and degradation.

Cleavage of Target mRNA

AGO2 Endonuclease Activity: Within the RISC, the AGO2 protein acts as an endonuclease to cleave the target mRNA. The endonucleolytic cleavage occurs at a specific site between the 10th and 11th nucleotides relative to the 5' end of the guide strand.

mRNA Degradation Pathway: Following AGO2-mediated cleavage, the target mRNA fragments are rapidly degraded by cellular exonucleases. This degradation prevents the mRNA from being translated into protein, effectively silencing the gene.

Intracellular Dynamics of siRNA and RISC

Cytoplasmic Action: The siRNA-RISC complex primarily operates within the cytoplasm, targeting cytoplasmic mRNAs for degradation. However, intriguing evidence suggests that siRNA-loaded RISCs might shuttle between the cytoplasm and nucleolus, indicating a broader functional role, potentially impacting nuclear RNA processing events.

Nucleolus Association: Studies have indicated that RISCs associated with the nucleolus might participate in yet unidentified processes, potentially involving the regulation of various RNA species within the nucleus. This dynamic localization underscores the multifaceted roles of siRNAs beyond mere mRNA degradation.

Origins and Integration of siRNA Pathways

Endogenous vs. Exogenous siRNAs: siRNAs can originate from both endogenous and exogenous sources. Endogenous siRNAs are derived from cellular dsRNA, such as transposons or inverted-repeat transgenes, whereas exogenous siRNAs are introduced experimentally or therapeutically, such as synthetic siRNAs used in research or clinical settings.

Integration with Other RNAi Pathways: The siRNA pathway integrates seamlessly with other silencing pathways involving miRNAs and piwi-interacting RNAs (piRNAs). They share common processing enzymes like Dicer and Argonaute proteins, allowing a unified regulatory network to control gene expression at multiple levels.

In summary, siRNAs are short, double-stranded RNA molecules that mediate RNA interference, a biological process that downregulates gene expression. siRNAs are typically 21-23 nucleotides in length and are designed to be complementary to specific mRNA sequences. When introduced into a cell, siRNAs integrate into the RISC. The RISC uses the siRNA as a guide to identify and bind to complementary mRNA transcripts. This binding triggers the degradation of the target mRNA, thereby preventing its translation into protein.

The efficacy of siRNA-mediated gene silencing is largely attributed to this high specificity. The siRNA-RISC complex ensures that only the mRNA molecules with perfect or near-perfect complementarity to the siRNA are degraded, minimizing off-target effects and enhancing the accuracy of gene regulation.

Major Functions of siRNAs

Gene Silencing and Regulation

Post-Transcriptional Gene Silencing: siRNAs act as critical regulators by silencing specific gene expression at the post-transcriptional level. This silencing is achieved through the RNAi pathway, where siRNAs guide the RISC to degrade complementary target mRNA. The specificity and efficiency of this gene silencing make siRNAs invaluable in both physiological and experimental contexts.

Role in Cellular Defense: One of the primary natural functions of siRNAs is to serve as a defense mechanism against foreign genetic elements such as viruses and transposons. By targeting and degrading viral RNA or the mRNA of transposable elements, siRNAs help maintain genomic integrity. This intrinsic role underscores their importance in protecting organisms from infectious agents and maintaining genomic stability.

Research Applications

Functional Genomics: siRNAs are powerful tools in functional genomics, enabling researchers to selectively knock down the expression of individual genes. By observing the resulting phenotypes, scientists can infer the roles and interactions of specific genes, leading to a deeper understanding of biological pathways and cellular functions. This approach has been instrumental in identifying gene functions in various model organisms, from C. elegans to human cell lines.

High-Throughput Screening: The specificity of siRNA-mediated gene silencing makes them ideal for high-throughput screening (HTS) in drug discovery and genetic research. Researchers employ siRNA libraries to systematically silence thousands of genes, allowing for the identification of critical genes involved in disease processes, potential drug targets, and pathways that can be modulated for therapeutic interventions.

Modeling Diseases: By silencing disease-related genes, siRNAs are used to create in vitro and in vivo models of human diseases. These models are essential for studying disease mechanisms, testing drug efficacy, and understanding the genetic basis of various conditions. For example, siRNAs targeting oncogenes or tumor suppressor genes help create cancer models, providing insights into tumor biology and potential therapeutic strategies.

Therapeutic Potential

Targeting 'Undruggable' Genes: Many genes encode proteins that lack suitable binding sites for conventional drugs, making them 'undruggable' by traditional small molecules. siRNAs can target virtually any mRNA, allowing the inhibition of gene expression for these traditionally undruggable targets. This ability opens new therapeutic avenues for conditions previously deemed untreatable with existing pharmaceutical approaches.

Cancer Therapy: siRNA therapeutics hold significant promise in cancer treatment by targeting oncogenes and pathways involved in tumor growth, metastasis, and drug resistance. Current clinical trials are exploring siRNAs targeting genes such as PLK1, RRM2, and VEGF:

TKM-080301 targets polo-like kinase 1 (PLK1), a cell-cycle protein essential for mitosis, and is in phase 1/2 clinical trials for neuroendocrine and adrenocortical cancers.

CALAA-01 targets the M2 subunit of ribonucleotide reductase (RRM2), using a transferrin-receptor-targeting delivery system, aimed at treating solid tumors.

ALN-VSP02 suppresses both kinesin spindle protein (KSP) and vascular endothelial growth factor (VEGF), targeting advanced solid tumors with liver metastasis.

Genetic Disorders: siRNA-based therapies offer potential treatments for genetic disorders by silencing mutated or malfunctioning genes. For instance, siRNAs are being investigated for conditions like hemophilia and hereditary amyloidosis, where reducing the expression of specific defective genes can alleviate disease symptoms.

Viral Infections: Targeting viral RNA, siRNAs can inhibit viral replication, providing a novel antiviral strategy. siRNAs designed against conserved regions of viral genomes can potentially suppress a wide range of viruses, including those responsible for significant global health challenges such as HIV, hepatitis, and respiratory syncytial virus (RSV).

Mechanisms of Action in Gene Regulation

mRNA Cleavage and Degradation: The primary mechanism by which siRNAs regulate gene expression is through the cleavage and degradation of target mRNA. The guide strand of the siRNA directs the RISC to the complementary mRNA, where the AGO2 protein cleaves the mRNA. The resulting mRNA fragments are then degraded by cellular exonucleases, effectively silencing the gene.

Feedback and Homeostasis: siRNAs also play roles in cellular feedback mechanisms and maintaining homeostasis. By fine-tuning the expression of essential genes, siRNAs help cells adapt to environmental changes and stressors. This regulatory function is vital for processes such as cell cycle control, apoptosis, and differentiation.

Off-Target Effects and Minimization Strategies

Challenges with Off-Target Effects: While siRNAs are highly specific, they can sometimes induce off-target effects by partially binding to non-target mRNAs with similar sequences. These unintended gene silencing events can lead to undesirable phenotypic consequences and complicate data interpretation in research applications.

Strategies to Minimize Off-Target Effects: Researchers employ various strategies to mitigate off-target effects, including:

Chemical Modifications: Modifying siRNA molecules, such as 2'-O-methylation, can enhance specificity and reduce immune activation.

Careful Design and Validation: Using computational tools and extensive validation processes to design siRNAs minimizes off-target interactions and optimizes target specificity.

Pooling siRNAs: Using pools of siRNAs targeting different regions of the same mRNA can reduce the off-target effects by diluting the chance of unintended interactions.

Applications Beyond Gene Silencing

Epigenetic Regulation: Recent evidence highlights that siRNAs can impact gene expression through epigenetic mechanisms, including alterations in chromatin structure and DNA methylation. These findings suggest that siRNAs have a more extensive role in gene regulation, influencing cellular processes beyond their direct effect on mRNA degradation.

Synthetic Biology and Genetic Engineering: In synthetic biology and genetic engineering, siRNAs are employed as precise tools to regulate gene expression in engineered organisms and cellular systems. This level of control is essential for the development of gene circuits, biosensors, and various biotechnological applications, enabling tailored modifications to genetic frameworks.

siRNA vs miRNA

To learn more, please refer to "How to Distinguish between siRNA, shRNA, and miRNA".

siRNA vs RNAi

To learn more about RNAi, please refer to "RNAi: Mechanisms, Applications, and Comparative Insights"

Figure 1 Gene silencing mechanisms of siRNA and miRNA.

Next-Generation Sequencing in siRNA Detection

siRNA technology has revolutionized the field of genetic research and therapeutic development. By harnessing the power of RNAi, siRNA sequencing has become a pivotal method for elucidating gene function, understanding regulatory networks, and developing targeted therapies.

This article provides an in-depth exploration of siRNA sequencing, covering its principles, methodologies, applications, and advantages.

Principles of siRNA Sequencing

siRNA sequencing is a sophisticated technique that leverages the principles of RNAi to explore gene function and regulatory networks. The following sections detail the core principles of siRNA sequencing, including the mechanism of action, library preparation, and the impact of siRNA molecules on gene expression.

Library Preparation for siRNA Sequencing

The initial step in siRNA sequencing involves the preparation of a comprehensive siRNA library. This library comprises a diverse array of siRNA molecules, each targeting different genes or gene regions. The process typically begins with the design and synthesis of siRNA molecules. Advanced high-throughput synthesis platforms allow researchers to generate large-scale siRNA libraries efficiently.

Once synthesized, the siRNAs are validated for their ability to specifically target their intended mRNAs. This validation is crucial to ensure the accuracy and effectiveness of the subsequent sequencing and analysis. The validated siRNA library is then used to transfect target cells, enabling researchers to study the functional effects of gene silencing.

Transfection and Functional Impact

Transfection is the process of introducing siRNA molecules into target cells. Various methods can be used for transfection, including lipid-mediated transfection, electroporation, and viral delivery systems. The choice of method depends on the cell type, the nature of the siRNA, and the specific experimental goals.

After transfection, cells are cultured under conditions that allow the siRNA molecules to exert their effects. The duration of culture and the concentration of siRNA can influence the extent of gene silencing and the resultant phenotypic changes. Researchers monitor these changes to assess the functional impact of the siRNAs on gene expression and cellular processes.

Sequencing and Analysis of Gene Expression

Following siRNA treatment, RNA is extracted from the cells for sequencing. RNA-seq is employed to obtain a comprehensive profile of gene expression. This technique involves converting the extracted RNA into cDNA, which is then sequenced to generate a detailed expression profile.

The sequencing data are analyzed to identify differentially expressed genes—those whose expression levels are significantly altered due to siRNA treatment. Bioinformatics tools are used to compare expression profiles before and after siRNA treatment, allowing researchers to determine the specific genes targeted by the siRNAs and their roles in cellular pathways.

Insights into Gene Function and Regulation

By integrating siRNA sequencing data with functional assays and bioinformatics analyses, researchers can gain valuable insights into gene function and regulatory mechanisms. This approach facilitates the identification of key genes involved in various biological processes and diseases. For instance, siRNA sequencing has been instrumental in uncovering gene networks associated with cancer progression, neurodegenerative diseases, and immune responses.

Applications of siRNA Sequencing

siRNA sequencing is a transformative tool in molecular biology, providing insights into gene function, gene regulation, and cellular processes. Its applications span a wide range of research areas and therapeutic developments. The following sections detail the key applications of siRNA sequencing, highlighting its role in functional genomics, drug discovery, disease modeling, and personalized medicine.

Functional Genomics and Gene Discovery

siRNA sequencing is instrumental in functional genomics, enabling researchers to dissect gene functions and regulatory pathways. By systematically silencing genes and analyzing the resulting phenotypic changes, scientists can identify the roles of individual genes in various biological processes. This approach helps in uncovering:

• Gene Function: Determining the specific functions of genes within complex cellular networks. For instance, siRNA screening can reveal the involvement of certain genes in cell cycle regulation, apoptosis, or metabolic pathways.
• Gene Interactions: Exploring gene interactions and identifying potential gene regulatory networks. This can elucidate how different genes collaborate to influence cellular behavior or disease progression.

Drug Discovery and Development

In the pharmaceutical industry, siRNA sequencing accelerates drug discovery and development by facilitating target identification and validation. The key contributions include:

• Target Validation: Identifying and validating novel drug targets by assessing the effects of siRNA-mediated gene silencing on disease-related pathways. For example, siRNA screening can confirm the involvement of specific genes in cancer cell proliferation, guiding the development of targeted therapies.
• Mechanism of Action: Understanding the mechanisms of action of potential drug candidates. siRNA sequencing helps in elucidating how drugs affect gene expression and contribute to therapeutic outcomes.

Disease Modeling and Therapeutic Research

siRNA sequencing plays a crucial role in disease modeling by simulating disease conditions and studying the molecular basis of diseases. Applications in this area include:

• Disease Mechanisms: Investigating the molecular mechanisms underlying diseases by silencing genes associated with specific conditions. For instance, researchers use siRNA to model neurodegenerative diseases and study the impact of gene silencing on neuronal health.
• Therapeutic Strategies: Developing and testing new therapeutic strategies. By targeting disease-associated genes with siRNA, researchers can evaluate the efficacy of potential treatments and optimize therapeutic approaches.

Personalized Medicine

siRNA sequencing contributes to the field of personalized medicine by enabling tailored therapeutic approaches based on individual genetic profiles. Applications include:

• Patient-Specific Therapies: Designing personalized treatments by identifying genetic variants that influence drug response. siRNA sequencing can help in understanding how individual patients' genetic make-up affects their response to specific drugs.
• Biomarker Discovery: Identifying biomarkers for disease diagnosis and prognosis. siRNA sequencing aids in discovering biomarkers that are indicative of disease states or responses to therapy, improving diagnostic accuracy and treatment efficacy.

Functional Characterization of Non-Coding RNAs

siRNA sequencing extends beyond protein-coding genes to explore the functional roles of non-coding RNAs. This includes:

• miRNAs: Investigating the impact of siRNA-mediated inhibition of specific miRNAs on gene expression and cellular functions. This approach can reveal how miRNAs regulate gene networks and contribute to disease processes.
• lncRNAs: Exploring the functions of lncRNAs and their roles in gene regulation and cellular processes. siRNA sequencing helps in understanding how lncRNAs influence gene expression and contribute to disease pathology.

Validation of RNAi-based Therapeutics

siRNA sequencing also supports the validation and optimization of RNA interference-based therapeutics. This includes:

• Therapeutic Optimization: Fine-tuning RNAi-based therapies to enhance their specificity and efficacy. By analyzing the effects of different siRNAs, researchers can optimize therapeutic strategies to achieve better outcomes.
• Off-Target Analysis: Assessing and minimizing off-target effects of siRNA therapies. siRNA sequencing helps in identifying unintended gene silencing effects and refining siRNA design to reduce off-target interactions.

Advantages of siRNA Sequencing

siRNA sequencing represents a powerful tool for gene function analysis, target identification, and functional genomics. By leveraging the principles of RNA interference and advanced sequencing technologies, researchers can gain valuable insights into gene regulation and disease mechanisms. As the field continues to evolve, siRNA sequencing will remain a critical component of genomic research and therapeutic development.

CD Genomics offers cutting-edge siRNA sequencing services, providing researchers with the tools and expertise needed to advance their studies and drive scientific discoveries.

References:

1. Lam JK, Chow MY, Zhang Y, Leung SW. siRNA Versus miRNA as Therapeutics for Gene Silencing. Mol Ther Nucleic Acids. 2015
2. Dana H, Chalbatani GM, Mahmoodzadeh H, Karimloo R, Rezaiean O, Moradzadeh A, Mehmandoost N, Moazzen F, Mazraeh A, Marmari V, Ebrahimi M, Rashno MM, Abadi SJ, Gharagouzlo E. Molecular Mechanisms and Biological Functions of siRNA. Int J Biomed Sci. 2017
3. Niu X, Sun Y, Chen Z, Li R, Padmanabhan C, Ruan J, Kreuze JF, Ling K, Fei Z, Gao S. Using Small RNA-seq Data to Detect siRNA Duplexes Induced by Plant Viruses. Genes (Basel). 2017