2. 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)

3. 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

5. 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

6. 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

7. 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

8. 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

10. 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

11. 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

13. 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

14. Eukaryotic gene expression without intervention by a Morpholino

15. 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

16. 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

17. Splicing blocked by a Morpholino oligo

18. 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

19. 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

20. 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

21. Case Study

22. 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

23. 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