RNA interference:
By M A R T GunaratnaIndex No: 8060
Special Degree in Bioinformatics
A molecular evolutionary perspective towards strengthening its blossoming applications
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Why the emphasis on RNA?• Molecules involved in the mechanism of
RNAi are non-protein - coded RNAs transcribed
from ‘junk DNA’• Biogenesis of miRNAs (microRNAs) that
are involved in:- Cell proliferation- Cell differentiation- Cell development- Programmed cell death- Interactions between viruses and host cells- Regulates activity of 1/3 of human protein-encoding genes
• Solution for C-value paradox / enigma
JUNK DNA TURNED OUT TO BE GOLD!!!!
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Nuclear Membrane
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C. elegansPetunias
Short hairpin RNA
siRNA with 3’ overhangs D. melanogaster
Craig Mellow & Andrew Fire Nobel prize 2006
Physiology or Medicine
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Plasma Membrane
RNAi blossoming applicationsDrug discovery
Therapeutic applications
Cancer
Neurodegenerative diseases
Duchenne Muscular Dystrophy & Haemophilia
Age-related macular degeneration (AMD)
• Research purposes• Stempeutics • Medical diagnostics
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Neurodegenerative Diseases
AMD
Cancer
http://stemcell.taragana.net/wp-content/uploads/2008/10/stem-cell-therapy-india1.jpg 20.06.2009http://www-tc.pbs.org/wgbh/nova/sciencenow/3210/images/02-cure-cancer.jpg 20.06.2009http://www.myvisiontest.com/img/upload/Macular_degeneration.jpg20.06.2009
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Organs for which RNAi proof of concept has been demonstrated
Challenges in RNAi Efficacious delivery
Stability
Specificity
Potency
1010http://www.nature.com/gt/journal/v13/n6/fig_tab/3302654f2.html#figure-title 20.06.2009
Spurious specificity & lack of potency: causative agents
Erroneous siRNA designing algorithms leads to Brute-Force
search of siRNAs ● Activation of immune responses
Constraints in developing
longer siRNAs● Complicated
synthesis
Target sequen
ce diversit
y
● Dive
rsify
ing
or
posi
tive
● Purif
ying
or
nega
tive
● Neut
ralSelection pressure
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Analysis of Darwinian selection pressure
dN - Number of sites with non-synonymous substitutions
dS - Number of sites with synonymous substitutions
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ω = dN / dS
ω > 1 – Positive selectionω = 1 – Neutral selectionω < 1 – Negative selection Charles Darwin at the
age of 31…http://upload.wikimedia.org/wikipedia/commons/e/ed/Charles-Darwin-31.jpg20.06.2009
PAML - Phylogenetic Analysis by Maximum Likelihood
Rich repertoire of advance substitution models
Utility of maximum likelihood method
Facilitates understanding of molecular evolution
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Getting started…
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Translation
Alignment of In-frame
Removal of identical sequences
Tree generation
Codon substitution modelsCODEML program package
ü ω not averaged throughout the phylogenetic tree
ü Codon is considered the evolutionary unit
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Branch models
Site models
Branch – Site models
Facts to be considered before proceeding…Number of sequences > 6Tree length > 0.11Number of Sequences should not be highly similar or highly diverged
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Parameters in site models
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Model NSsites
p Parameters
M0 (one ratio) 0 1 ωM1a (neutral) [N]
1 2 p0 (p1 = 1 – p0), ω0 < 1, ω1 = 1
M2a (selection) [A]
2 4 p0, p1 (p2 = 1 – p0 – p1), ω0 < 1, ω1 = 1, ω2 > 1
M3 (discrete) 3 5 p0, p1 (p2 = 1 – p0 – p1) ω0, ω1, ω2 M7 (beta) [N] 7 2 p, q
M8 (beta&ω) [A] 8 4 p0 (p1 = 1 – p0), p, q, ωs > 1
NOTE: M0 and M3 are used only to measure the variability of ω among sitesp is the number of free parameters in the ω distribution.[A] – Alternative model [N] – Null model
Parameters in Branch-site models
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Site class
Parameters Background
ωForeground
ω
0 p0 0 < ω0 < 1 0 < ω0 < 1
1 p1 ω1 = 1 ω1 = 1
2a (1 – p0 – p1) * p0 / (p0 + p1) 0 < ω0 < 1 ω2 ≥ 1
2b (1 – p0 – p1) * p1 / (p0 + p1) ω1 = 1 ω2 ≥ 1
NOTE: Branch-site model A is specified using model = 2 NSsites = 2. This is the alternative model in the branch-site test of positive selection. The null model fixes ω2 = 1. The likelihood ratio test has df = 1 At a particular instance;Foreground branch – Branch analyzed for positive evolutionBackground branch – Branch not analyzed for positive evolution
Control file in CODEML
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seqfile = xxxxx.nuc
treefile = xxxxx.trees
outfile = mlcxxxxx * main result file name
model = 2 * models for codons:
* 0:one, 1:b, 2:2 or more dN/dS ratios for branches
NSsites = 8
fix_kappa = 0 * 1: kappa fixed, 0: kappa to be estimated
kappa = 1.6 * initial or fixed kappa
fix_omega = 0 * 1: omega or omega_1 fixed, 0: estimate
omega = .8 * initial or fixed omega, for codons or codon-based
AAs
ncatG = 10 * # of categories in dG of NSsites models
getSE = 1 * 0: don't want them, 1: want S.E.s of estimates
cleandata = 0 * remove sites with ambiguity data (1:yes,
0:no)?
method = 0 * 0: simultaneous; 1: one branch at a time
CODEML results
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tree length = 0.52544
Model 7: beta (10 categories) lnL(ntime: 17 np: 20): -2001.404646 +0.000000 dN/dS (w) for site classes (K=10)p: 0.10000 0.10000 0.10000 0.10000 0.10000 0.10000 0.10000 0.10000 0.10000 0.10000w: 0.00000 0.00000 0.00000 0.00000 0.00000 0.00000 0.00000 0.20940 1.00000 1.00000 Model 8: beta&w>1 (11 categories) lnL(ntime: 17 np: 22): -2000.054824 +0.000000 w= 1.88981 dN/dS (w) for site classes (K=11) p: 0.08895 0.08895 0.08895 0.08895 0.08895 0.08895 0.08895 0.08895 0.08895 0.08895 0.11047w: 0.03624 0.04694 0.05420 0.06050 0.06653 0.07272 0.07947 0.08741 0.09800 0.11736 1.88981
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CODEML results contd…
Significance of results
Hypothesis Testing
●LRT = 2● Chi – square (
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Posterior probabilities of
● NEB
● BEB
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Local optima and the global optimum
Result
MEC – Mechanistic Empirical Combination Model
Assimilates empirical amino acid substitution probabilities
Selecton server at http://selecton.tau.ac.il/
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Commercial potential of RNAi
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Global market estimates for RNAi;
Year 2010: $ 3.5 billion
Year 2015: $ 10.5 billion• Major areas of RNAi applications in the expected
market;
• Drug discovery and research• Potential therapeutics• miRNA mediated diagnostics
Impact of molecular evolutionary analysis on advancement of RNAi
2626http://www.nature.com/nrn/journal/v6/n6/images/nrn1688-i1.gif 20.06.2009http://img.medscape.com/fullsize/migrated/548/891/ncpn548891.fig2.jpg 20.06.2009http://www.pbs.org/wgbh/nova/sciencenow/3210/images/02-cure-hepatitis.jpg 20.06.2009
Clues for universal therapeutics and diagnostics
Assurance of efficacious activity
Accelerate extensive researches on RNAi
Saves time, resources, labor and money in designing siRNAs & identifying potent miRNAs
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development of blue rosesSuccess: observed…
First brain stem cell therapySUCCESS: PENDING…???
http://www.stem-cells-news.com/1/2009/05/first-stem-cell-brain-surgery/ 20.06.2009http://3.bp.blogspot.com/_Ha_cQO3YwFc/R6kGRjT5LiI/AAAAAAAADoQ/oi80O5KGIy4/s400/blueroses.jpg 20.06.2009
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THANK YOU
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