AAPPLICATION OFPPLICATION OF

MMOLECULAROLECULAR B BIOLOGY IOLOGY TTECHNIQUESECHNIQUES

IN IN AASTROBIOLOGYSTROBIOLOGY

RICCARDO S. GATTA Ph.D. Molecular Genetics, I.C.G.E.B. New Delhi, Reg’d Pharmacist

JULIAN CHELA-FLORES The Abdus Salam International Centre for Theoretical Physics Strada Costiera 11; 34136 Trieste, Italy and

Instituto de Estudios Avanzados, Caracas 1015A, Venezuela

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IINTRODUCTIONNTRODUCTION

CLASSIC MOLECULAR BIOLOGY APPROACH TO

RECOGNISE MOLECULAR BIOSIGNATURES,

APPLY THIS IN AN ISOLATED ENVIRONMENT.

&

SELECT TARGET,

SELECT TECHNIQUE,

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HAEKEL (1834-1919)

WOESE(1980’s)

SSEARCH FOREARCH FOR L LIFE IN THE IFE IN THE UUNIVERSENIVERSE

DARWIN (1809-1882)

ANCESTOR

MODERN

??

KNOWN

UNKNOWN ??

ANAXIMANDER (VI BCE)DEMOCRITUS (460-370 BCE)

The Origin

OBSERVATIONS - EVOLUTION

MACROSCOPIC

MICROSCOPIC

MOLECULAR

DIVERGENTCONVERGENT

EVOLUTIONMODELS OF

PARALLEL

LAST

ANCESTOR

UNIVERSAL

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Yes

EEVOLUTION VOLUTION && NNATURALATURAL S SELECTIONELECTION UNIVERSAL CONCEPTS?

No

continue…

LIFE…?

(…and skip next slide)

IF

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Structure

LIFE AS A TARGET

IDENTIFY & QUANTIFY

Complex StructuresChemistryEmergent Chirality

LIFE

Complexity

Energy

Transduction

Polymers

ReplicationSpecific InteractionsDriving ForcesMolecular / Cellular

Metabolism More ComplexityMore DisequilibriumWaste

Yes

EEVOLUTION VOLUTION & N& NATURAL ATURAL SSELECTIONELECTION

UNIVERSAL CONCEPTS?

No

continue…

LIFE…?

(…and skip next slide)

IF

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Figure from Labfrontier Corporation

REPLICATION:

DNA ↕

DNA

TRANSLATION:

mRNA↓

Protein

TRANSCRIPTION:

DNA ↓

mRNA

CCENTRALENTRAL D DOGMAOGMA OFOF M MOLECULAOLECULARR B BIOLOGYIOLOGYCrick, 1958

DO NOT

code for :

DNA RNA PROTEINS

“… to apply only to present-day organisms, and not to events in the remote past, such as the origin of life or the origin of the code.” Crick, 1970

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K v

K ir Ca v Na v

Early K channels

Voltage-gated channels

PHANEROZOIC

570 Myo - present

PROTEROZOIC

2.5 Gyo – 570 Myo

ARCHEAN

4.6 – 2.5 Gyo

~800 million

years ago

Ancient Proteins, membrane channels

TIME /

EVOLUTIONModern Proteins

Example of divergence

from LUCA at

Check-Point IntervalsCheck-Point Intervals

For:

Homology with known molecules

Na

Ca

K

EEVOLUTIONARY VOLUTIONARY CCRITERIARITERIA

Moczydlowski, 1998

Homology with known molecules

Where can we seek homology?

BUT

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1:

2:

Homology in:

BLUE 2, Joan Miro’.

1: INDIVIDUAL ELEMENTS

COMPOSITION 8, Vassily Kandinsky.

2: BUILD UP (CONSTRUCTION)

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2: BUILD UP (CONSTRUCTION)

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DECONSTRUCTIDENTIFY BASIC ELEMENTSEUROPA

FROM: NASA, JPL, UA, UC.

3: THE PARADIGM: 2: BUILD UP (CONSTRUCTION)

FIRSTTHEN

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SSAMPLE AMPLE AANALYSISNALYSIS

ASSESS ENVIRONMENTAL CONDITIONS,

LEGITIMISE FURTHER ASSAYS,

DIRECT CHOICE OF PLAUSIBLE ASSAYS,

INDICATE OPTIMAL CONDITIONS FOR ASSAYS.

ENVIRONMENTAL SAMPLE ANALYSIS

PRELIMINARY ANALYSIS

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MMOLECULAROLECULAR T TARGETSARGETS

DNA HIGHLY CONSERVED MOTIFSRNA TATAAA, Shine-Dalgarno, rRNA sub-types, shifts in pyrimidine/purine ratios…

TRANSLATIONAL - ENZYMATIC messenger RNA, inhibitory RNA…

Protein STRUCTURAL SCAFFOLDSOther: conserved charged residues, essential enzymes, functionally divergent… superfamilies traceable to LUCA…

chirality…

special fatty acids or sugar residues…

lipids, peptidolipids, lipopolysaccharides,

conserved charged residues, essential enzymes, functionally divergent… superfamilies traceable to LUCA…

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PRIMER

TaqmanRApplied Biosystems

MMOLECULAROLECULAR R RECOGNITION ECOGNITION TTECHNIQUESECHNIQUES

PCR

DNA

POLYMERASE CHAIN REACTION

PROBE

FRAGMENT

RESTRICTION FRAGMENT LENGTH POLYMORPHISM

RIBOTYPING of rDNA (5S, 16S, 23S etc. rRNA genes)PHYLOGENETIC STUDIES, DIAGNOSTICS…

RIBOSOMAL GENE SEQUENCES

R F L P

RFLPs

LARGE FRAGMENTS (1,000 – 20,000 kb)

FRAGMENT

2D gel

PULSE FIELD GEL ELECTROPHORESISP F G E

SMALLER FRAGMENTS (10 – 1000 kb)RELATIONSHIP - BACTERIAL STRAINSRESOLUTION IN 2D

WHOLE GENOME

From: Hernandez-Rivas and Scherf, 1997

GENOME SIZE – PHYLOGENETIC STUDIES…

From: Pillai SD

BA

↓

FLUORESCENCE IN SITU HYBRIDISATION

VERSATILE TECHNIQUE

S HF I

National Human Genome Research Institute (NHGRI)   Darryl Leja  

DENATURE &

LABEL WITH FLUORESCENT DYE

HYBRIDISE

PULSE FIELD GEL ELECTROPHORESISRESTRICTION FRAGMENT LENGTH POLYMORPHISM

FLUORESCENCE IN SITU HYBRIDISATION

PROBE

SINGLE NUCLEOTIDE POLYMORPHISMS (SNP)EXPRESSED SEQUENCE TAGS (EST)

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SEQUENCING

MMOLECULAROLECULAR R RECOGNITION ECOGNITION TTECHNIQUESECHNIQUES

DNA ONCE THE TARGET SEQUENCE IS IDENTIFIED

RT-PCR

RNA

REAL TIME REVERSE TRANSCRIPTION PCR

DIFFERENTIAL DISPLAY REVERSE TRANSCRIPTION PCR

SINGLE CELL mRNA RESOLUTION

RNA EXPRESSION PROFILES

NORTHERN BLOT

WESTERN BLOT

SOUTHERN BLOT

↓↓ ↓

BLOTTING

↓proteins

Protein PROBES: fluorescent technology

From: Alberts, Bray, Johnson, Lewis, Raff, Roberts, Walterc 1999

ANTIBODIES, mono-poly…

green fluorescent protein…

labeling environment…

limited number of conformations…CLICKCLICKtoto

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Nikon Eclipse TE2000 Gatta, ICGEB New Delhi

MOL-BIO LAB USE:

AS A LIGHT MICROSCOPE: for cell counting, etc.

IN FLUORESCENCE MODE: to highlight transfection efficiency...

MINIMAL SAMPLE PREPARATION,

BASIC TECHNIQUE,

APPLICATIONS MAY BE LIMITLESS...

FLUORESCENCE TECHNIQUES – IMAGE ENHANCING

MetaMorph® Universal Imaging Corp.Olympus Inc.

Differential Interference Contrast (DIC), (optic ganglion tissue from rat retina)

Fluorescence illumination: -diazonium salt fluorochrome (fast blue).

Phase Contrast, (3T3 fibroblast monolayer, NIH)

Fluorescence illumination: -4',6-diamidino-2-phenylindole (DAPI),

Remove some ‘out-of-focus’ effects:

Digital deconvolution.

- phase rings on fluorite objective.

AANALYTICAL NALYTICAL DDEVICESEVICESFLUORESCENCE TECHNIQUES

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Streptomyces lividans K+ channel (KcsA), Moczydlowski, 1998.Aplysia californica K+ channel (Kv), Miller, 2000.

Recognitio

n

Site A ?

Recognition

Site B ?

Intra-Cellular

Extra-Cellular

HOW CAN THEY BE RECOGNISED?IION ON CCHANNELSHANNELS ASAS T TARGETSARGETS

ANTIBODIES

RATIONAL DESIGN

From: Miller, 2000.

WHOLE / LYSED CELLS

, DIABODIES , BIOMIMETIC PROBES…CLICKCLICK

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AANALYTICAL NALYTICAL DDEVICESEVICES Potential for Size and Power Optimisation

(Figure: Bruker BioSpin AVANCETMICE® )

(Figure: Bruker BioSpin Minispec® )(Figure: Haskin Research Group)

NMR

“Hyphenation” with other techniques:

…other devices:

Confocal Microscope,

Chromatography (CZE),Spectroscopy (MS, IMS).

UnityNOVA Varian Inc.

Teepe et al., 2001 RamanIMS

SNOM/ATM, Seiko Instruments, Inc SPM200TM WITek

From: MBARI.

From: JPL

MS

CEMicro-Tech Scientific, Inc.

AANALYTICAL NALYTICAL DDEVICESEVICESPotential for Size and Power Optimisation

Scanning Near-Field Optical Microscope, Atomic Force Microscope.

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SNOM/ATM

AANALYTICAL NALYTICAL DDEVICESEVICES

From: MBARI.

From:Wang J, NMSU

LABORATORY-ON-A-CHIP

Gourley and Sasaki, 2001

From:Wang J, NMSUGast and Fiehn, 2003

GENERAL STRUCTURE:CE

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“Hyphenation” with other techniques:

Scanning Near-Field Optical Microscope, Atomic Force Microscope.

…other devices:

Confocal Microscope,

Chromatography (CZE),Spectroscopy (MS, IMS).

Teepe et al., 2001 IMS

From: MBARI.

From: JPL

MS

CE

Potential for Size and Power Optimisation

µ-Capillary Electrophoresis Ion Mobility Spectrometer

macromolecule separation.

field investigation of VOCs,

chip-based : ITP / CZE, combination with detection : - electrochemical (Φ, Ω, MS etc.), - optical (diode-LIF), - for analysis of sample / environment.

Applications:

multi-organic component analysis,

AANALYTICAL NALYTICAL DDEVICESEVICESFOR A PRELIMINARY ANALYSIS

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(Figure: ©1998 by Alberts, Bray, Johnson, Lewis, Raff,

Roberts, Walter Published by Garland Publishing.)(Figure: GeneChip® Affymetrix)

Specific Analysis:Few Observations =

more hypotheses

Multi-Protein Analysis : More Variables =

More DATA

AANALYTICAL NALYTICAL DDEVICESEVICESMICRO-ARRAYS / BIO-CHIPS

ES

Bio-Chip “reading” techniques:

choice of techniques and materials, automation and miniaturisation,

- antibodies / fluorescence…

- cantilever biosensors,

Courtesy of Flavio Robles / PDB

NanoReaderTM BioForce NanoSciences

Daniel T. Colbert

Silica from Tethya aurantia, Weaver J. and Morse D

- novel detection methods,

- surface plasmon resonance,

Figure: Biacore® Corporation

Biacore®

- new materials…

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SSUMMARYUMMARY

REVIEWED AVAILABLE TECHNIQUES,

RATIONALE OF TARGET SELECTION,

SELECTION OF ANALYSIS TECHNIQUES.

APPLICATION TO ASTROBIOLOGY PARADIGM,

FFUTUREUTURE C CHALLENGESHALLENGES

MICROFLUIDIC ENVIRONMENT FOR ASSAYS,

PERMIT MULTIPLE SIMULTANEOUS ASSAYS (tens of 1000’s),

SEQUENTIAL NANO-MODELING OF PROBES,

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AACKNOWLEDGEMENTSCKNOWLEDGEMENTS

Participants of the Seventh Trieste Conference on Chemical Evolution and the Origin of Life 

the the abdus salam abdus salam       international centre for theoretical physics      international centre for theoretical physics

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