NANOPROBE FOR LIVE CELL GENOMICS

Kumar Wickramasinghe

COLLABORATORS

• Kavita Arora, PhD –

• Rahul Warrior, PhD –

• Ed Nelson, MD –

• Arthur Lander, MD, PhD –

Development and Cell Biology, UCI

Development and Cell Biology, UCI

Chief, Hematology and Oncology, UCI

Development and Cell Biology, UCICenter for Complex Biological Systems

We introduce a unique technology that can rapidly profile gene expression levels within a living cell at unprecedented sensitivity - down to single molecules.

Capability opens up

New methods for diagnosis of genetic diseases

New analysis instrument in cell biology for tracking the fate of single cells in response to external stimuli such as drugs, silencing or signaling molecules

New analytical tool that is likely to have significant impact in cell biology

OVERVIEW

Current technology uses DNA arrays to profile gene expression levels

DNA arrays typically require mRNA extracted from thousands to millions of cells for analysis

Averaging over thousands of cells reduces sensitivity and masks subtle variations in gene expression

The ability to detect low level fluctuations in gene expression in living cells – down to single molecules – is key to detecting genetic mutations that result in diseases and for studying the effects of drugs or signaling molecules

NEEDS THAT ARE NOT MET BY CURRENT TECHNOLOGY

STOCHASTIC TRANSCRIPTION

Average accurately represents the population

Average hides information about the population

Cells randomly express high or low levels

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Adapted from: MacNeil L T , Walhout A J Genome Res. 2011

STOCHASTIC TRANSCRIPTION AND PHENOTYPE

Raser and O’Shea, 2005

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Fingerprints from identical twins

Genetically identical cloned cats

Stochastic distribution of cell fates in insect photoreceptors

Losick and Desplan, 2008

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f’ = -dk

object

BACKGROUND OF THE TECHNOLOGY

Force – Vibrating Mode Atomic Force Microscope - AFM

Light – Apertureless Near-Field - sSNOM, ANSOM, TERS

Heat – Scanning Thermal Microscope - SThM

Magnetism – Scanning Magnetic Force Microscope - MFM

Electrostatics – Electrostatic Force Microscope - EFM

Electrochemical Potential – Scanning Kelvin Probe Microscope - SKPM

Photovoltage – Scanning Photovoltage Microscopy - SPVM

SCANNING PROBE MICROSCOPYNanoscale Measurements of…..

AFM MOLECULAR SORTING AND DELIVERY(Unal, Frommer, Wickramasinghe, Appl. Phys. Lett. 88, 183105, 2006)

SCHEMATIC FOR MOLECULAR SAMPLING WITHIN A SINGLE CELL

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Nanoprobe tip modification mRNA extraction from a cellby dielectrophoretic force

Selective mRNA hybridization

mRNA collectionmRNA quantification

Direct and selective sampling of mRNA from a single cell or an array of cellsusing a nanoscale probeNo laborious and expensive chemical purification steps necessarymRNA is copied into cDNA and then quantified using standard qPCR

PLATFORM CONCEPT

PROOF OF CONCEPTmRNA from a single living cell has been selectively extracted and quantifiedCopy numbers down to a few molecules has been detected for selected mRNA species (HPRT)Multiple mRNA species have been detected at the same timeTransfected breast cancer genes were detected through expression analysis in single cells

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10- fold dilutions ofGAPDH mRNAHPRT mRNA

“Targeted Profiling of Breast Cancer Gene in a Single Living Cell”, Analytical Biochemistry, 2010

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Time (mins)

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miRNA quantification of hsa-mir-125a during apoptosis

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10 fold dilution ofnegative control

100 fold dilution ofnegative control

Breast cancer gene (rat neu -ve)

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x mRNA molecules

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Breast cancer gene (rat neu +ve)

EPIGENETIC CHANGES CONTRIBUTE TO CANCER GENE MISEXPRESSION

OFF ON

Zhou et al (2011), 12; 7‐18

1.Testis2. PCR negative control3. Electrophoresis ON4. Electrophoresis OFF5. Whole cell skewer6. cDNA water control

1 2 3 4 5 6Tubulin 5

1 2 3 4 5 6BRCA1

DETECTION OF EPIGENETIC ALLELE SPECIFIC EXPRESSION

• Large single cell - 0.5 mm long • Can be manipulated under non-sterile conditions• Robust system for microinjections• Rapid development, 6000 cells in 2 hours, larva hatches in 24 hours• Sensitive phenotypic assay for effects on development and viability • Maternal mRNA pool not replenished during development

DROSOPHILA DEVELOPMENT IS A POWERFUL IN VIVO ASSAY

Sophisticated transgenic tools that allow dynamic visualization of GFP-tagged bcd mRNA in vivoExtensive array of genetic mutations, gene duplications, etc., to aid in calibration and quantitationGenomic data from the ModEncode project

Tracking mRNA Expression in Space and Time

a b c

CapabilityOur technology

Microfluidics technology

Live cell transcriptomics Yes NoReagent volume 1 ul 50 ulDisease detection Yes NoIn situ pathology Yes NoSpeed 10 secs 1-2 hrsComplexity Simple ComplicatedCost Lower HigherChemical purification steps No YesProtein assays Yes NoSingle cell transfection Yes NoLive cell response Yes No

COMPARISON WITH EMERGING TECHNOLOGIES

Electric Field ONmRNA collection

Electric Field OFFmRNA DEPOSITION

WHERE WE WANT TO BE ……

semi-automated mRNA extraction platform

BACKUP

NorthernMicroarrayReal-time PCRRNA-seq

FISH

MS2-GFP

SCA

Require averaging of gene expression over many cells

Can follow expression in vivo, but requires genetic modification, challenging to quantitate

Can analyze individual cells, but is not dynamic

In vivo, quantitative, single cell analysis, that can be followed over time

COMMON METHODS FOR RNA QUANTIFICATION

TRANSCRIPTIONAL PROFILING OF CANCER CELLS

Gunn, Nelson, Journal of Biomedical Materials Research, 2010

(c)

DIFFERENTIAL mRNA LOCALIZATION IN NEURONS

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DISRUPTION OF mRNA LOCALIZATION IN NEURODEGENERATIVE DISEASES

Detect and monitor transcriptional responses at the single cell level over time

Determine operating parameters relating to sensitivity, spatial resolution, and number of different transcripts that can be assayed simultaneously

Develop methods to maximize sensitivity without affecting the developmental program

Calibration of mRNA extraction procedure

Develop an SCA based system for delivery of siRNAs and proteins for single cell analysis

BROAD GOALS