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Bunnell lecture
inhibition of gene expression
| Heading | Oligos Antisense | RNA Antisense | Ribozymes | siRNA |
|---|---|---|---|---|
| Structure | Short, complementary NA strands | ssRNA | Antisense w/ catalytic activity | dsRNA & dicer; si Rna from long Ds; miRNA from short 70 NT stem loop RNA hairpin; nuclease that cuts dsRNA strand; loss of dicer leads to loss of silencing; doesn’t create intermediates; found in drosoph, c elegans, mammals, & plants ; RISC RNA complex |
| Inhibition mechanism | Binds target mRNA forms dsNA blocks translation promotes degradation via RNAases | Forms RNA duplex degraded by RNAse H or blocked by translation | Binds target and hydrolyzes its backbone | Dicer cleaves dsRNA to 21-23 bp siRNAs siRNA activates RISC RISC cleaves mRNA |
| Degraded? | within cell | Yes | No | Yeah, in cytoplasm |
| Notes | tech used by anti-sense drugs;inherently unstable ; put phosphorothioate oligos or stem loop structure to increase stability | alternative to oligos; production of antisense is intracellular | Hammerhead: U-H; Hairpin: C-U-G; 1st found in gp 1 self splicing intron & rna-ase @ P enzyme; ie: plants, bacteria, protozoa, animal, & fungi | 19-21 BP duplex w/ 1-2 overhangs;cleaves 12nt from siRNA 3’ ; injecting sense & anti-sense together inhibits gene more than either alone; used for: 1. gene fxn determination; 2. study pathways;3. identify & validate targets; 4. generate KO models |
| PTGS(post transc gene silencing) | found in plants | takes active transgene leads to aberrant dsRNA siRNA targeted degradation | transgenic & endogenous RNA | -**conserved mechanism of gene regulation from plants to mammals** |
| Delivery | 1. in vitro active uptake of molecules, varies by cell type; 2. in vivo very inefficient uptake via liposomes | -direct introduction or production in cells & tissues ; microinjections into cells;plasmids; not practical – bc too many cells | Same as RNA antisense??? | Need Large Amts of RNAi; 1. direct dsRNA injection;2. transfection: only transient effect ; 3. expression vectors: plasmid based use polIII promoters; 4. expression of sense & antisense: different polIII promoters |
| Disadv | -nonspecific effecgts; cytotoxic at hi conc | retarded one | (blank) | (blank) |
| Factors affecting effectiveness | 1. IC conc of oligs (delivery & stability); 2. [mRNA target] ; 3. mRNA production & turnover rate ; 4. interaction rate | 1. requires hi expression levels of antisense RNA; 2. not practical in vivo | - single molecule so lower levels needed | - lo conc min non-specific effects ;- specificity verified via testing indep siRNAs against same target; - ds target must be @ < 26-32 nt;- no introns & promoters ; - no specific sequence required |
| Conclusions | (blank) | (blank) | (blank) | powerful for analyzing unknown genes in sequenced genomes ; general applications in mammalian cells |
| Selecting the target | (blank) | (blank) | (blank) | find 21 nt that begins w/ AA because SI w/ UU overhang best ; 2-4 target sequences w. 30-50% GC content ; negative control with same NT composition, but no sequence homology; -positive control of additional siRNAs w/ same target |
| 4 reasons to inhibit gene expression (in general) | find Gene/Protein role | Analyze P-P interactions | 3) role in organ function | 4) Tx apps: - primarily @ infections diseases (HIV, HBV, TB) |
| Molecular Mechanisms of Anti-sense: | - inhibit mRNA splicing (splice donor/splice acceptor) | - translational arrest | - disrupt necessary rna structure | - destabilization of rna by inhibiting 5-cap or polyA |
| Measure effectiveness of oligs | - northern blot or RT-PCR to see gene inhibition | - DR curves (varies cell lines & delivery) | - Assess multiple oligo chemical classes;- Perform time course to asses potency | - Evaluate specificity & toxicity |
| the universal goal is: | SILENCE genes through Watson-Crick base pairing | (blank) | (blank) | (blank) |
Created by:
nidhers
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