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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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