Single molecule mechanical analysis

Many flavors of single molecule techniques

Force

Electrical

In silico

now

Three most popular methods to measure single molecule forces

AFM

Magnetic

tweezers

Optical

tweezers

4

Ashkin et al., Opt. Lett. 11, 288 (1986)

Explaining optical trap: ray optics

Scattering force: F_scat

gradient force: F_grad

gradient force: F_grad

Light refraction (twice) causes light momentum change.

By Newton’s third law, bead experience momentum change in opposite direction 🡪 gradient force, bringing the bead toward focus

Light reflection pushes the bead away 🡪 scattering force

When gradient force > scattering force 🡪 You have an optical trap!!

Tethering geometries

Visscher et al. Nature (1999)

Nature, News and Views (2009)

Wuite et al., Nature (2000)

1. Surface-based (kinesin)

2. Micropipette-based (DNAp)

3. Two-trap (viral DNA packaging)

But there are many others...

Flexible DNA

~ 30 µm

Flow stretching turned off at t=0

Time t

Optically

trapped

bead

DNA as a Worm-Like Chain

Bustamante, Smith, Marko, Siggia

Freely Jointed Chain

Model Fit

Worm-Like Chain

Model Fit

Force-extension curve

Persistence length

~ 50 nm for dsDNA

About 150 bp

​

1-3 nm for ssDNA

Or about 3 nt.

Worm Like Chain (WLC)

RNA polymerase backtracking

Long pause

backtracking

Shaevitz et al, Nature (2003)

Force Ramp

Constant Force

Two experimental formats

Hairpin see also: Woodside et al., PNAS (2006)

Streptavidin bead

3’ biotin

3’ digoxigenin

Anti-digoxigenin bead

DNA handle

DNA handle

Hairpin

Stretching a DNA “hairpin”:

Example: Force Ramp

13

Hairpin protocol: Woodside et al., PNAS (2006)

Red = stretching

Green = relaxing

Qi et al., eLife (2013)

Example: Force Ramp

Example: Constant Force

Krishna Neupane et al. Science 2016;352:239-242

Zoom poll

Three most popular methods to measure single molecule forces

AFM

Magnetic

tweezers

Optical

tweezers

AFM pulling experiment: polyprotein unfolding

Three most popular methods to measure single molecule forces

AFM

Magnetic

tweezers

Optical

tweezers

Magnetic Tweezers and DNA

Can be conveniently used to stretch and twist DNA.

• DNA is stretched more if magnet is moved down.
• DNA also tends to twist if twist magnets

(since m follows B).

(either mechanically, or electrically move magnets)

​

Forces ranging from a few fN to nearly 100 pN: a huge Range

Induced dipole moment m ∝ B (magnetic field).

The force is upward, i.e., where B is highest.

With Super-paramagnetic bead, no permanent dipole.

Magnetic potential energy U = - mB ∝ - B².

F = - ∇ U (Force is directed toward energy minimum)

Magnetic tweezers to manipulate supercoiling state of DNA