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Gene Silencing by using morpholino molecules
Submitted to the Dr. S. B. Verulkar Prof & Head, Dept of Plant Mol Biology & Biotech Indira Gandhi Krishi Vishwavidyalaya, Raipur (C.G.), INDIA MBB 601- Advance in Plant Molecular Biology Kakade Datta Pandurangrao PhD Scholar (Plant Molecular Biology and Biotechnology)
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History of gene silencing
Gene silencing; Process that results in down-regulation/ suppression of a gene at the RNA level 1990- Petunias- Firstly called “co-suppression” Overexpression of chalcone synthase in petunias unexpectedly resulted in white petunias 1992- Mold (Neurospora crassa)- The introduced gene led to inactivation of the mold's own gene in about 30% of the transformed cells. They called this gene inactivation “quelling” 1998- Worm (C. Elegans) - Fire and Mello; 2006 Nobel Prize in Physiology & Medicine
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Branches of Genetics study
Forward Genetics Begins with a mutant (altered) phenotype and addresses the question, “what is the genotype?” or mutation in which gene caused the alteration in the phenotype. Reverse Genetics Begins with the mutant gene sequence and asks the questions “what is the resulting effect on the phenotype?” Find the possible phenotypes that may derive from a specific genetic sequence
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Techniques for gene silencing
RNA interference technology Virus induced gene silencing Targeted gene disruption by homologous recombination Insertional mutations/ T-DNA insertions Transposons mediated mutations Fast neutron based insertions Chemical mutation (Tilling/Eco-tilling) Morpholino based
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Morpholino based Morpholino-molecules are non-ionic nucleic acid analogs. Sometimes referred to as “PMO” (Phosphorodiamidate Morpholinos Oligo) Possess altered backbone compared to nucleic acid Morpholino oligos are a class of antisense, that block access of other molecules to specific sequences within nucleic acids Morpholinos block small (~25 base) regions of the base-pairing surfaces of ribonucleic acid (RNA) Most Morpholinos are used as research tools for reverse genetics by knocking down gene function
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Structure Morpholinos are synthetic molecules that are the product of a redesign of natural nucleic acid structure Usually ~25 bases in length Bind to complementary sequences of RNA by standard nucleic acid base-pairing Structural difference between Morpholinos and DNA is that, Morpholinos have standard nucleic acid bases, which are bound to morpholine rings instead of deoxyribose rings Linked through phosphorodiamidate groups instead of phosphates Morpholinos in organisms or cells are uncharged molecules
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Advantages of Morpholino oligos over DNA and RNA-based knockdown reagents...
Very robust and stable molecules due to non-biological backbone Specificity at low concentrations Largely free of off-target effects Steric block mechanism allows for additional applications, e.g. block of miRNA maturation or alteration of mRNA splicing Properties of Stability, Nuclease-resistance, Efficacy, Long-term activity, Specificity
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Functions Morpholinos do not degrade their target RNA molecules unlike many antisense structural types (e.g., phospho-rothioates, siRNA) Act by “steric blocking”, binding to a target sequence within an RNA and simply getting in the way of molecules that might otherwise interact with the RNA Morpholino oligos are often used to investigate the role of a specific mRNA transcript in an embryo
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Gene expression in eukaryotes
pre-mRNA is transcribed in the nucleus, introns are spliced out, then the mature mRNA is exported from the nucleus to the cytoplasm The small subunit of the ribosome usually starts by binding to one end of the mRNA with various other eukaryotic initiation factors, forming the initiation complex The initiation complex scans along the mRNA strand until it reaches a start codon, and then the large subunit of the ribosome attaches to the small subunit and translation of a protein begins Morpholino can modify splicing or block translation, depending on the morpholino’s base sequence
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Eukaryotic gene expression without intervention by a Morpholino
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Blocking translation Morpholino bound to the 5'-untranslated region of messenger RNA (mRNA), Morpholinos can interfere with progression of the ribosomal initiation complex from the 5' cap to the start codon This prevents translation of the coding region of the targeted transcript (called "knocking down" gene expression) Translation blocked by a Morpholino oligo
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Modifying pre-mRNA splicing
Morpholinos can interfere with pre-mRNA processing steps either by a)Preventing splice-directing small nuclear ribonucleoproteins (snRNP) complexes from binding to their targets at the borders of introns on a strand of premRNA, b) Or by blocking the nucleophilic adenine base and preventing it from forming the splice lariat structure, c) Or by interfering with the binding of splice regulatory proteins such as splice silencers
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Splicing blocked by a Morpholino oligo
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Other Applications: Blocking other mRNA sites, use as probes, etc.
Morpholinos have been used to block miRNA activity and maturation Fluorescein tagged Morpholinos, used as probes for in-situ hybridization to miRNAs Morpholinos can block ribozyme activity Morpholinos can block editing of RNA
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Specificity, stability and non-antisense
effects Morpholinos have become a standard knockdown tool in animal embryonic systems Morpholino oligo’s shows sequence-specificity and lack of non-antisense effects Because of their completely unnatural backbones, morpholinos are not recognized by cellular proteins, nucleases do not degrade morpholinos nor are they degraded in serum or in cells
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Delivery Morpholino to be effective, it must be delivered past the cell membrane into the cytosol of a cell Different methods are used for delivery into embryos, into cultured cells or into adult animals Microinjection Electroporation Endo-Porter peptide (which causes the Morpholino to be released from endosomes) Liposome delivery
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Case Study
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Ekker experiment in Zebrafish…
For blocking of expression of the GFP (Green Fluorescent Protein) Injected fluorescently labeled morpholino oligonucleotide into sphere-stage of the zebrafish embryo along with the controlled oligomer Morpholino and oligomer both are complementary to the start codon of GFP Morpholino blocks the expression of the GFP but oligomer did not Level of GFP mRNA was not changed Shows that the RNase H is not involved in the mechanism Which proved that morpholine only blocks the target mRNA and which was not degraded further
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References Jasmine HP Chan, Shuhui Lim and WS Fred Wong, Antisense oligonucleotides: from design to therapeutic application, Clinical and Experimental Pharmacology and Physiology (2006) 33, 533– 540 David R Corey and John M Abrams, Morpholino antisense oligonucleotides: tool for investigating vertebrate development, Genome biology 2001,2(5):reviews Jon D. Moulton and Shan Jiang, Gene Knockdowns in Adult Animals: PPMOs and Vivo-Morpholinos, Molecules 2009, 14, Sarah L. DeVos & Timothy M. Miller, Antisense Oligonucleotides: Treating Neurodegeneration at the Level of RNA, Neurotherapeutics, DOI /s
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