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sirna and mirna
- 1. IMPORTANCE OF siRNAAND miRNA Presented by:- Vignesh M 1stsem M Pharm Pharmacology Dept GCP Bangalore
- 2. Contents to discuss Introduction Importance of non coding RNA’s and its classification siRNA synthesis, pathway and its importance miRNA pathway and its importance Difference between siRNA and miRNA
- 3. INTRODUCTION Gene expression The behaviourof a cell is determined not only by what genes it inherits but also by which of those genes are expressed at any given time. The flow of genetic information in cells generally proceeds from DNA to RNA to protein.
- 4. Enhancers and Silencers Theseare DNA Elements Located at Variable Distances from the Promoter Enhancer – stimulate gene transcription also called as transcriptional activators Silencers – inhibits transcription also called as transcriptional repressors Insulators - restrict the influence of enhancers and silencers
- 5. Genes are regulatedat either transcriptional level or post transcriptional level Transcriptional Gene Silencing Post Transcriptional gene silencing Genomic imprinting Antisense RNA technology Paramutation RNAi Technology Transposon silencing miRNA Transgene silencing shRNA Position Effect siRNA RNA directed DNA methylation
- 6. Coding RNA’s CodingRNA’s can be defined as that sequence of nucleic acids which can be transcribed to proteins. The release of a draft sequence of the human genome, in 2001 by International Human Genome Sequencing Consortium predicted that protein-coding genes contributed just 1.5% of the entire sequence! remaining was non protein coding RNA’s.
- 7. Non coding RNA’s Non-coding RNAs, commonly defined as transcripts that are not translated into proteins, exist in simple as well as in complex forms of life. The complexity of organisms is not just dependent on the number of protein-coding genes but their non-coding RNAs, too. Encyclopedia of DNA Elements (ENCODE) consortium concluded that 93% of bases were of non-coding RNAs. Only a small fraction of these transcripts were mRNAs, which encoded proteins of considerable length.
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- 9. The concept ofRNA interference RNA interference or gene knockdown is the process in which double-stranded RNA is cleaved by a RNA’s III-type enzyme called Dicer into small interfering RNA duplex. This then directs sequence-specific, homology dependent, post transcriptional gene silencing by binding to its complementary RNA and triggering its elimination through degradation or by inducing translational inhibition. In 1998, Fire and Mello published a scientific breakthrough by injecting single- stranded sense and antisense RNAs together into C. elegans, they were able to interfere with the expression of selected genes, in a very effective and specific manner
- 10. siRNA Small interferingRNA (siRNA) is a short double-stranded RNA 21-to-23 nucleotides in length. It behaves as a mediator of the RNA interference phenomenon by silencing a specific gene expression by the cleavage of a target mRNA at the post-transcriptional level in the cytoplasm. siRNA can be produced invitro by using synthetic DNA template containing the T7 RNA polymerase promoter region followed by the needed RNA sequence. This sequence can be produced using a DNA synthesizer and then amplified by a PCR.
- 11. Properties of siRNA Naked siRNA has low stability when administered intravenously in biological system. Degrades rapidly. Possesses poor cellular uptake. Lack of simple and efficient delivery systems. The major limitation for the use of siRNA, both in vitro and in vivo, is the inability of naked siRNA to passively diffuse through cellular membranes.
- 12. Approaches to improveits properties Nanosized cationic delivery systems have been studied which are based on polyelectrolyte complexes resulting from the electrostatic interaction between the negatively charged siRNA and cationic polymers or lipids. Direct conjugation of aptamers, lipids, peptides, proteins, or polymers to siRNA can improve its in vivo pharmacokinetics, increase half-life, and increase delivery efficiency.
- 13. siRNA Pathway Exogenous double-strandedRNAs (dsRNAs) served frequently as precursors, which were observed to be processed into siRNAs duplexes of 21 bp and with two-nucleotide 3 overhangs.
- 14. Importance of siRNA 1.Rolein immunity RNAi plays a more general role in innate immune defense of higher animals. During viral infection, mammals rely on an early detection of foreign ribonucleic acids to mount a rapid antiviral response. The cells co-ordinating the initiation of this antiviral response sense viral RNAs through Toll-like receptors (TLRs) or retinoic acid inducible gene I (RIG-I) like receptors. Plasmacytoid dendritic cells and macrophages/monocytes are the main detectors of TLR7/8 agonists amongst other immune blood cells.
- 15. 2. siRNA inNasopharyngeal Carcinoma The specific silencing of hyaluronan receptor (CD44) gene expression by RNAi in the nasopharyngeal carcinoma cell line (CNE-2L2) resulted in profound reduction of malignant potential of the cells, including growth, in vitro colony formation, tumorigenesis, and metastasis of tumors in mice. Direct injection of the adenoviruses harboring and producing siRNA to CD44 into the tumor inoculated with CNE-2L2 cells in mice caused inhibition of tumor growth.
- 16. Tumour growth inhibitedby CNE- 2L2 Normal Gene pool Mutation Mutant Gene Mutant mRNA Synthetic siRNA Knockdown of Mutant Gene Altered Protein Alleviation of disease/disorder Pathogenesis Progression of disease/disorder
- 17. 3. siRNA inthe treatment of Asthma An siRNA-based therapy targeting the transcription factor GATA3 in activated T helper cells subtype 2 (TH2 cells), one of the key upstream factors involved in asthma Targeting mRNA coding for GATA3 with siRNA could enable post- transcriptional gene silencing of the transcription factor which is overexpressed in activated TH2 cells.
- 18. Sequence of events explainingpathogenesis of asthma
- 19. 4. siRNA activityin Dendritic cells Invivo Delivery of siRNA into Dendritic cells for the purpose of immune modulation finds some use CD40 siRNA was incorporated within the interior of 86 nm liposomes, which were decorated with surface-bound mAb NLDC-145 as a targeting mechanism. Dendritic cells (DCs) are the most potent stimulators of T cell activation. Paradoxically, DCs also possess immune regulatory properties that play an essential role in preventing autoimmune disease by inducing antigen-specific tolerance to self-antigens. In vitro CD40-silenced DCs significantly inhibited the proliferation of alloreactive T cells in an MLR. We have previously generated tolerogenic DCs (tol-DCs) by silencing immune-related genes in the field of functional genomics.
- 20. Delivery can beenhanced either by improving siRNA attributes through chemical modifications of the duplex by Using different formulations such as cationic liposomes or polymers. Encapsulation of a stabilized siRNA within a liposomal nanoparticle greatly enhances serum half-life and bioavailability. liposomal and polymeric nanoparticles coated with targeting ligands have been used for delivery. Thus, a combination of these methods to generate stabilized siRNA encapsulated nanoparticles may provide a method of choice to enhance delivery.
- 21. 5. siRNA inNeurodegenerative Disorders Specific siRNA knockdown of mutant SOD 1 slows amyotrophic lateral sclerosis (ALS) in animal models. Chemically synthesized siRNAs targeting the gene responsible for Huntington’s disease provide a therapeutic benefit in an animal model of the human disease. These improved effects included reduction in neuronal pathology and an improvement in motor behavior. The RNAi therapy reduced expression of mutant huntingtin in the brain and sustained a benefit in motor behavior for at least one week.
- 22. miRNA MicroRNAs (miRNAs)are a class of short, 21-24 nucleotide endogenously- initiated non-coding RNAs that post-transcriptionally control gene expression via either translational repression or mRNA degradation. miRNAs play significant roles in regulatory mechanisms operating in various organisms, including developmental timing and host-pathogen interactions as well as cell differentiation, proliferation, apoptosis and tumorigenesis.
- 23. The miRNA Pathway microRNAs(miRNAs) are short RNA species expressed in most eukaryotes and are known as key regulators of gene expression that act through imperfect base pairing with their target messenger RNA (mRNA).
- 24. Importance of miRNA 1.Cleavage of HIV-1 proteins by miRNA miRNA is known to repress mRNA translation through recognition of specific binding sites usually located in their 3 ¢ untranslated region thereby inhibiting translation initiation. This anti-HIV-1 therapy is one of the RNA-based strategies that include antisenses, ribozymes and aptamers. Some of them are currently being tested in clinical trials.
- 25. Although RNAi-based antiviraltherapies are promising, HIV-1 has been shown to escape RNAi induced by a specific siRNA. In these cases, the emergence of mutants was observed, either showing nucleotide substitutions or deletions within the targeted sequence.
- 26. 2. miRNA TargetedGlioma Associated Antigen-1 protein. More than half of the human miRNA genes are located at sites known to be involved in cancers, such as fragile sites, minimal regions of loss of heterozygosity, minimal regions of amplification or common breakpoint regions. We engineered Gli-1 miRNA to down regulate protein expression. Autonomous activation of the Gli-1/2 pathway provides metastatic tumor cells with a means of efficiently proliferating at a distance from continually Shh-expressing epithelium.
- 27. Because Glifunction is a last and essential step of the Shh pathway, its inhibition may inhibit Shh signaling at any level. We found that our engineered Gli-1 miRNA and Gli-1 small temporal/duplex miRNA significantly inhibited the proliferation and division of the tumor cells.
- 28. 3. miRNA inLung Cancer Detection we designed a nanogenosensor based on cysteamine-capped gold nanoparticles (Cys - AuNPs) that makes it possible to detect the miRNA (miR-25) related to lung cancer. A single stranded probe (ss-probe) was covalently immobilized on the surface of a Cys-AuNPs/glassy carbon electrode (GCE) by glutaraldehyde (GA), and then the ss-probe was hybridized with the lung cancer-specific miRNA. The preparation process of the proposed electrochemical nanogenosensor was characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS).
- 29. The analyticalperformance of this nanogenosensor in detecting miR-25 was investigated. It could significantly make a distinction between a fully matched miRNA strand and a miRNA strand comprising a single-base mutation. Furthermore, this nanogenosensor is capable of detecting miR-25 directly in human blood plasma without using PCR or any other amplification methods
- 30. 4.miRNA in TumourSupression miRNAs are encoded in the genome and are transcribed by RNA polymerase II (pol II) as long precursor transcripts, which are known as primary miRNAs (pri-miRNAs). The pre-miRNA is recognized by the nuclear export factor Exportin-5, which transports it to the cytoplasm which uses Ran-GTP as a co-factor. Further cytoplasmic processing by a second RNase III enzyme named Dicer performs a second cleavage to generate double-stranded 18- to 24- nucleotide long miRNA.
- 31. One of thesetwo strands the guide strand is incorporated in an ATP independent manner into the RNA-induced silencing complex, which includes as core components the Argonaute proteins (Ago1–4 in humans). Only one strand of the degree of complementarity between an miRNA and its target determines the mechanism of binding and silencing.
- 32. Difference between siRNAand miRNA Properties siRNA miRNA Origin Created from double stranded RNA templates by the enzyme DICER Distinct genomic loci. Encoded by their own genes. Discovered 1999 1993 Argonaute reqired ABO1, ABO4, ABO6, ABO7 ABO1, ABO10 Mechanism of gene regulation Transcriptional as well as post transcriptional Post transcriptional only Biogenesis Long bimolecular RNA duplexes or extended hairpins Single RNA molecules that include an imperfect stem loop secondary structure Nature of target Mediate the silencing of the same genes from which they originate Regulate different genes
- 33. Targeting SiRNA tendto base pair perfectly with their target miRNA have imperfect base pairing Interaction SiRNA-RISC complex interact with 5’end of their target transcript miRNA-RISC complex interact with 3’end of their target Size 21-25 Nucleotides 20-22 Nucleotides Functions Defence against transposons, viruses and stress adaption Cell development and cell differentiation, regulation of development processes, biotic and abiotic stress responses Complementary with target Pratially or fully complementary Fully complementary Evolutionary conservation Rarely conserved in related organism Always conserved in related organism
- 34. References: 1. Molecular pharmacologyfrom DNA to drug discovery by John Dickenson, Fiona Freeman, Chris Lloyd Mills, Christian Thode, Shiva Sivasubramaniam. 2. RNA Interference From Biology to Clinical Applications by Abubaker M. E. Sidahmed, Bruce Wilkie, Wei-Ping Min, Thomas Ichim. 3. siRNA and miRNA Gene Silencing From Bench to Bedside by Shigeru Takasaki. 4. Small Non-Coding RNAs Methods and Protocols by Mathieu Rederstorff. Articles: 1. Journal of Dental Research (siRNA, miRNA, and shRNA: in vivo Applications) by P .N. Pushparaj, J.J. Aarthi, J. Manikandan and S.D. Kumar. 2. Analytical Methods (Application of cysteamine-capped gold nanoparticles for early detection of lung cancer-specific miRNA (miR-25) in human blood plasma) by Amineh Asadzadeh-Firouzabadi, Hamid R. Zare.
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