Bionano Chemistry Lab

Muhammad Ehsan(2013652616)

Date: 2014-06-03

Molecular Imaging …

DNA/Protein

MicroArrays

Source:

Uzgiris, GE CRD

Tjuvajev, Blasberg, Larson, MSKCC

Biochemistry

Pharmacology

MOLECULAR

IMAGING

Molecular

Biology

PET, MRI, MRSI

Imaging Physics,

Engineering

Affymetrix

Pull together an Interdisciplinary Approach

Various Imaging Techniques

• Magnetic Resonance Imaging (MRI)

• Positron Emission Tomography (PET)

• Single-Photon Emission Computed Tomography (SPECT)

• Fluorescence Resonance Energy Transfer (FRET)

• Fluorescence

• Bioluminescence

Magnetic Resonance Imaging (MRI)

• Used to visualize the inside of living organisms

• Demonstrates pathological or other physiological alterations of living tissues (i.e. tumors)

• Uses radio frequency signals to acquire images

• Based on the relaxation properties of excited Hydrogen nuclei in water

http://en.wikipedia.org/wiki/Image:User-FastFission-brain.gifhttp://en.wikipedia.org/wiki/Image:3Dbrain.gif

Positron Emission Tomography (PET)• A nuclear medicine imaging technique

that produces a 3D image or map of functional processes in the body

• Uses a short-lived radioactive tracer isotope which decays by emitting a positron (has been chemically incorporated into a metabolically active molecule) and is injected into the living animal, usually in the blood

• Commonly used alongside CT scans or MRI scans, giving both anatomic and metabolic information

http://en.wikipedia.org/wiki/Image:PET-MIPS-anim.gif

Single-Photon Emission Computed Tomography (SPECT)

• A nuclear medicine tomographic imaging technique using gamma rays

able to provide true 3D information

– A 2D view of the 3D distribution of a radionucleotide from

multiple angles

• A computer is used to apply a tomographic reconstruction algorithm to

yield a 3D dataset

– Can be manipulated to show thin slices along any chosen axis of

the body

Fluorescence Resonance Energy Transfer (FRET)

• Energy transfer mechanism between two fluorescent molecules

• Useful tool to quantify molecular dynamics in biophysics, such as protein-

protein interactions, protein-DNA interactions, and protein conformational

changes

– Monitors the complex formation between two molecules, one is labeled

with a donor and the other with an acceptor, which are then mixed

– When they dissociate, the donor emission is detected upon the donor

excitation, but when together, the acceptor emission is predominant

Fluorescence• Production and emission of light by a living organism as the result of a

chemical reaction during which chemical energy is converted to light energy

– Uses an external light source with a low-pass filter to excite the fluorescent molecules

• Green Fluorescent Protein, originally found in the Aequorea victoriaspecies of jelly fish

– Been biochemically modified to produce Green, Yellow, Blue, Cyan, and Red Fluorescent Proteins for use in various research techniques using a reporter

• Limited by tissue autofluorescence, as well as the light being able to first get into the living model and sensing the target fluorescent molecule, then having that fluorescence get back out of the model and to the detector (a lot of scattering occurs)

http://wwwchem.leidenuniv.nl/metprot/armand/images/029l.jpghttp://en.wikipedia.org/wiki/Image:Aequorea_victoria.jpg http://www.upenn.edu/pennnews/photos/704/mice.jpg

Bioluminescence

• Bioluminescence is the production and emission of light by a

living organism.

• Bioluminescence occurs widely in marine vertebrates

and invertebrates, as well as in some fungi, microorganisms

and terrestrial invertebrates.

• Some symbiotic organisms carried within larger organisms

produce light

Cherry et al. 2004

Autofluorescence

Cells contain molecules, which become fluorescentwhen excited by UV/Visual radiation of suitablewavelength.

This fluorescence emission, arising from endogenousfluorophores, is an intrinsic property of cells and iscalled auto-fluorescence which is different fromfluorescent signals obtained by adding exogenousmarkers.

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Applications

• Fluorescent proteins have become valuable tools for biomedical

research,

• as protein tags, Reporters of gene expression,

biosensor components, and cell lineage tracers

Goals of in vivo AFPs measurements

• Measuring molecular distances• Detecting conformational

changes• Detecting interactions• Localizing interactions• Following interaction dynamics• Reporting enzymatic activities

and intracellular conditions

Tracking Messengers

Small molecules as second messengers play a central role in signal transduction

.

One such ubiquitous messenger is nitric oxide (NO), which is involved in

various physiological and pathophysiological processes.

Diaminofluoresceins and diaminocyanines have been described as small

molecule-based sensors for the detection of NO.

However, these molecules do not sense NO directly but react with oxidized NO

products to yield a highly fluorescent product.

Probes for Nitric Oxide

• A fluorescent sensor that allows for the first time a direct detection of NO

was recently introduced by Lippard and colleagues.

•

Fluorescein-based sensor (CuFL) for detecting NO

www.acschemicalbiology.org/VOL.2 NO.1 • 31–38 • 2007

VOL.2 NO.1 • 31–38 • 2007

Creating Fluorescent Sensors for Enzymatic Activities by Design

• Fluorescein derivatives (dubbed TokyoGreens) (12) as sensors for β-galacto

sidase activity.

www.acschemicalbiology.org/VOL.2 NO.1 • 31–38 • 2007

• Rhodamine-based probes as sensors for esterase activity.

Esterases release the phenol of the so-called trimethyl lock group , and this

leads to rapid lactonization and liberation of the fluorophore

www.acschemicalbiology.org/VOL.2 NO.1 • 31–38 • 2007

Measuring Ions

• Zinc, although considered a trace element, is an abundant metal

ion whose concentration within eukaryotic cells is 100 µM.

• Zinc is bound to various proteins, including transcription factors,

and acts as a cofactor in several enzymes.

• The total concentration of zinc in cells is relatively high, whereas

the concentration of free or rapidly exchangeable zinc is very low.

• Estimates of the concentration of free zinc in prokaryotic cells are

in the femtomolar range.

• Measuring free or rapidly exchangeable zinc at picomolar or lower

concentrations in the presence of high concentrations of calcium

and magnesium thus requires a highly specific and sensitive

fluorescent sensor.

CA-based fluorescent sensor for measuring the concentration of zinc ions in living cells. CA is expressed as a fusion protein

with a cell-penetrating peptide (not shown) and labeled with the synthetic fluorophore AF594 at Cys36. Binding of zinc by

CA leads to subsequent binding of the fluorescent CA inhibitor dapoxyl sulphonamide (DS). Excitation of DS at 365 nm bo

und to CA results in efficient FRET to AF594 and emission at 617 nm. The measured emission at 617 nm upon excitation at

365 nm is standardized by dividing it by the emission at 617 nm measured upon direct excitation of AF594 at 540 nm.

Published in: Nils Johnsson; Kai Johnsson; ACS Chem. Biol. 2, 31-38.DOI: 10.1021/cb6003977Copyright © 2007

AFPs as sensors for biological processes face limitations.

First, AFPs are relatively bulky, in the best case monomers of 240 residues,

and size matters for all applications for which the distance between the AFP

and the activity to be recorded is critical.

Second, the spectral range of AFPs is limited. For example, no useful AFPs

are available in the near-infrared region, and different pairs of AFPs for

simultaneous FRET measurements in living cells have not yet been

established.

Third, for the construction of sensors for various crucial biomolecules and

enzymatic activities, AFPs offer no obvious solution.

Future outlook