Transcript Click here to presentation

Studying gene expression: An improved
high-content RNAi screening technology
Discovering more, faster, cost-effectively
Dr Asongwe L. A. Tantoh
Introduction
This presentation is intended to give an overview on:
•
a revolutionised high-content/throughput screening (HCS/HTS) platform
•
brief history on HCS/HTS inception
•
the different HTS platforms and their limitations
•
comparison between the different HTS platforms
•
our technology description and proof of concept of the platform
•
validation data from our high density microarray platform
•
other potential application(s) of the platform
•
the long term goal(s) behind the technology
Where it all started
Revolutionary events in functional genomics
1
Discovery of the RNAi mechanism
( Drs Andrew Fire and Craig Mello:2006)
RNAi mechanism
2
Sequencing of the Human genome (2000)
The human genome
Central Dogma of Molecular
Biology
RNAi mechanism
DNA replication
RNA
Transcription
DNA
RNA
Translation
Protei
Protein
n
Reverse
transcription
RNAi
mechanism
Folded with
function
Physiolog
y
Physiology
siRNA
Human genome
sequencing
Vast number of small molecule libraries encoding for the human genome
•
siRNA
•
miRNA
•
shRNA
•
CRISPR/Cas9; used for genome editing
Enable our understanding of how genes are regulated;
1. during biological processes
2. during pathogen-host interaction
How do we screen through hundreds of thousands
of chemical libraries with relative ease and in a short
time?
Traditional HT screening format:
Limitations
Well plate formats in high-density screening
384 well plate(25µl)
96 well plate (100µl)
1536 well plate (4µL)
•
expensive automated liquid handling devices required
•
fully automated integrated systems
•
huge infrastructure
•
highly trained workers required/ Experienced scientist
•
cost ineffective
Conventional HT microarray format:
Limitations
Conventional HT arrayers - like the one to the left:
•
are slow : 100 arrays in 17 hours
pins are washed, blotted and carry only 100nL
reservoir
•
are 95% wasteful of library print mixes
– mix viscosity leads to poor repeat prints (10-20
spots in one pass maximum)
– use print pins that are reused to print the array
•
print pins are expensive
•
Can not scale up
CSIR arrayer and its performance
•
cost effective
•
easy to operate
•
we measured printing quality using a single
continuous print of siRNA samples
•
our arrayer prints >95% spots in one contact
•
prints 500-750 essentially identical arrays
•
in 1.5 hours with 2 FTE
Our array technology
Our approach
conventional approach
The microarrayer pin used in current technology is
not a solution for HCS
capillary print element
•
•
•
•
•
•
low volume
very expensive (750$ each)
not suited to transfection mix
jams
very slow
wasteful of mix
Why capillaries?
•
capillaries deposit accurate spots
•
the reservoir volume of a capillary is 40X greater
than a print pin: 4000nL Vs. 100nL
•
capillaries don’t need refilling or washing
•
they fill automatically by capillary action
•
they exist in different sizes (from 150 to 600µm)
•
inexpensive
•
they are readily commercially available
Our platform: How we print
Step 3: stack the printing plates in the print head :
assembling the print array
Encapsulated siRNAs
Step 1: load the filler plate
with encapsulated siRNAs
1
4
Step 2: capillary loading
2
Step 4: contact print >1000 arrays
3
entire array 3 150 spots printed in one contact 2.5 Sec
scale: thousand plus copies
CSIR arrayer
Conventional
arrayer
CSIR arrayer vs
conventional arrayer
array
one shot
3 150 spots/2.5Sec
24
speed
1array in 2.5 Sec
1 array/hour
output
1000-1500 arrays
100 arrays
time
1.5 hours
15 hours
cost
<20 000$
>200 000$
waste
20%
95%
missing spots
<5%
6-10%
precision
25µm
similar
ease of use
****
*
Our microarray plate vs.
96, 384, 1 536 well plates
Number of 96 well plates
required/Persomics plate
Vol/well= 100ul (96 well plate)
32.8
8.2
2.05
High-density microarray plate
comprising of 3150 siRNA spots
High-density well plates
Number of 384 well plates
required/Persomics plate
Vol/well= 25ul (384 well plate)
Number of 1536 well plates
required/Persomics plate
Vol/well= 4ul (1536 well plate)
Reducing variation
HDS-plate
3 150 individual
experiments
less variation
96-well plates
required
Ease of use
HDS-plate
3 150 individual
experiments
Less handling
96-well plates
required
Our research
siRNA spot characterization
400
A
B
350
300
C
35
250
200
150
100
25
15
5
50
300
0
Capillary contact printing of encapsulated siRNA
•
•
•
Capillary contact printed optically addressable siRNA spot (A),
Confocal image, average diameter (B) and
Spot width distribution (C).
350
400
The HCS array principle
siRNA spot
each spot contains
an siRNA targeting a
specific protein
siRNA spot
cell cultivation
silencing
each spot silences
production of one protein
Verification of gene silencing
mean intensity/pixel .103
Suppression of NF-κB/p65 expression p65 siRNA
p65 siRNA spots
Creating wells without walls
Suppresses NF-kB/p65
expression
Control p56
siRNA
> 70% knockdown
in p65 expression
Verification of gene silencing
Arrest of cytokinesis by INCENP siRNA
Control siRNA spot
Creating wells without walls
Incenp siRNA spot
cells with multiple nuclei
Array data validation
TNF induced p65 translocation
+ TNF
unstimulated control cells
translocation into the nucleus
Assay conditions designed to give maximal p65 transport into the nucleus
p65 was detected in HeLa cells using indirect immuno-labelling after TNF stimulation
Array based screening
•
Arrays printed containing printed siRNA covering:
– all kinases, phosphatases, ubiquitinylases, proteases
– printed controls: control, XPO1, & p65 siRNA
• control siRNA is a non targeting siRNA silencing no genes
• XPO1 is the nuclear export factor for p65 & drives its recycling back out of the
nucleus
• p65 siRNA directly silences p65
• these controls enable understanding of the array quality in screening & XPO1
/p65 siRNA will give different phenotype to control
– XPO1 will accumulate p65 in the nucleus
– p65 will reduce p65 levels in the cells
•
Arrays comprise 3 150 individual siRNA spots
Knowledge directed pathway
exploration
•
we used two genes known to influence p65 nuclear transport to define where to find hits
– CHUK
– IKBKB
• both are kinases regulating signalling from TNF => p65/NFkB
• loss of these kinases stop nuclear import of p65
• both are in the printed library, as single spots on each array
•
32 replicates enable robust screening
– controls are well defined
– Principal Component analysis of the data gives excellent separation of controls, toxic
siRNA and CHUK/IKBKB
Basis used for hit selection
Genes connectivity results using
pathway studio
A
> 50% connectivity
Novel therapeutic targets
CHUK
IKBKB
XPO1
RELA
Non-targeting
B
Expressed phenotypes
Summary
•
we screened the TNF alpha pathway at the highest density ever performed
(to our knowledge)
•
100 800 individual siRNA cell based experiments were performed in 4 days,
with 2 extra days for analysis
•
high replicate, highly robust data
•
using conventional technology, this would require 3 months
•
CSIR/Persomics array technology...applied to cell screening
Commercialised platform
Persomics preparation lab
Boston
Persomics array printing lab
Arrays, opportunities
Beyond siRNA
•
siRNA is the most challenging format for HCS arrays
•
we want to move on to:
– compound arrays
– novel array architecture
•
compound arrays have great potential
•
since Bailey showed the principle in 2004, we are not
aware of any uptake of the method
Compound arrays
•
compound arrays have not been adopted because:
– technical complexity of making them with
arrayers
•
CSIR/Persomics arrayer was designed for mass
production of compound arrays
Proof of concept
Layer printing
POLYMER
2004 PNAS
PAO spot
PAO spot
DRUG
CELLS ON DRUGS
Bailey’s result
Bailey’s printing
approach
Single spot - localized drug effect
Phenylarsine oxide (PAO: toxic) + Ctrl spots
POLYMER
DRUG
drug impregnated in
polymer
PAO spot
PAO spot
Cells on drug
Bailey’s result reproduced at CSIR
Superior production method and higher effect
and reproducibility
CSIR compound
printing approach
Conclusion
MINIATURIZATION
3150 experiments
on one plate
CREATING A MINIATURIZED HIGH THROUGHPUT SCREENING PLATFORM
Conclusion: Our Goals
To offer South African and African researchers a high throughput screening product that is;
•
miniaturised and standardised for screening
•
commoditise the technology:
•
•
•
make it portable,
easy to manufacture
easy to use ‘out of the box’
•
exceed current technology standards
•
create outstanding products
Conclusion: Our Goals
To enable researchers access to rapidly test thousands of molecules in various bioassays in an easy
and timeous manner; i.e. screening through hundreds of;
• small interfering RNAs (siRNAs,miRNA,shRNA)
• Small-molecule compound
• drug Repurposing (alternative uses): FDA approved Drugs
• “cDNAs”
Using the current platform to screen for biological
targets that will serve as leads chemicals for new
therapeutic drugs
Thank you for your attention
Team Members:
Dr Dalu Mancama
Dr Lindiwe Thete
Dr Natasha Kolesnikova
Dr Alex Alexandre
Dr Hazel Mufhandu
Dr Asongwe Tantoh