Single Molecule Manipulation: Methods and Applications - Prof. C. J. Bustamante, Study notes of Physics

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Single Molecule Manipulation

Methods

Methods of Single Molecule

Manipulation

1873: The

Dutch

scientist

J.D. van der

Waals

proposes his

theory of continuity between the

gas and liquid states of matter

:

“All properties of matter depend on the strength and thedirection of the forces that molecules exert on eachother” 1889: Svante Arrhenius suggests that the rate of a

chemical reaction is determined by the rate ofattainment of a strained, high energy state or^ transition state

along its reaction coordinate

Bulk Methods vs. Single Molecule

Methods

Single Molecule Methods: •^

Molecules display a fast, instantaneous dynamics

-^

Behavior appear random and stochastic

-^

Fluctuations are predominant

-^

Molecules are seen to co-exist in various states. Populations are multimodal •^

Molecules can be found in states far from the mean of thepopulation (extreme states)

Why single Molecule Methods?

The microscopic view matters in describing the cell interior:Many cellular processes, such as:Chromosome replication and segregationDNA transcription, recombination, and RNA translationAre often carried out by a few molecules. Far from displayingsmooth dynamics these process are stochastic in nature.

Why single Molecule Methods?

The advent of methods of single molecule manipulation hasmade it possible, for the first time to:

Measure the forces that maintain the 3D structure of macromoleculesCharacterize the stress-strain relationships of moleculesMeasure the forces generated in chemical & biochemical reactionsInvestigate time-averaged and time-dependent fluctuationsCharacterize the dynamics of molecular motorsExert External forces and torques to alter the extent and fate of chem.Rxns.

Single Molecule Manipulation

Experiments

Two features distinguish single molecule manipulation

experiments

From their bulk counterparts:1)

The unique role played by forces or torques as directobservablesof the experiment

The importance of fluctuations

Together this area of research can be called:

Mechanochemistry

Technical Requirements

I)

Methods to locate molecules: a microscope that works in

liquids

a)

Optical: Fluorescence labelling, Particle tagging

b) Probe Microscopes: STM, SFM (AFM), SNOM

II) Means of manipulating or acting on single molecules:

a) Mechanical transducers: soft cantilevers, bendablemicropipettes

movable rigid micropipettes, etc

b) External Fields: Electric, magnetic and photon fields

III) Methods of spatial detection:

a) diffraction-limited displacement, centroid displacementb) optical lever detectionc) optical interference

Methods, Capabilities and Applications

Applications

Applications

Applications

Time-length and Force Scales of Experiments

Length Scale

The length scale is set by the type of process and the size ofthe molecules under study.

Range:

Angstroms (Small motions in a protein)Nanometers (Most processes)Microns (Stretching of a long DNA)

Single molecule manipulation instruments must be able tomake and /or measure displacements of nanometers or better

Force Scale

Forces can be:

InertialDissipative (frictional)Related to a potential of some sort

-On single molecule experiments, inertial forces are usuallynegligible compared to frictional forces: The low Reynoldsnumber regime.

F

v^ term

=

F γ

t^ accel

=

m γ

γ^

= fric. coeff

and m = sphere’s

mass

Expl: For a 1 micron silicabead in water, t

acc

is 10

s,

well below the time res. ofmost instruments.