Single Molecule Techniques

Question 1

What is the main goal of single-molecule techniques?

Answer 1

To study individual molecules directly, revealing mechanisms and heterogeneity hidden in ensemble measurements

Question 2

Why are single-molecule experiments important?

Answer 2

Ensemble averages mask the behaviour of individual molecules, whereas single-molecule studies can detect intermediate states, rare events, and mechanistic details

Question 3

What’s the main challenge in single-molecule studies?

Answer 3

Detecting extremely small signals above noise — requires sensitive detection and low background

Question 4

How dilute must samples be for single-molecule work?

Answer 4

Usually nanomolar concentrations (~10⁻⁹ M) to isolate single molecules

Question 5

What is optical trapping?

Answer 5

A technique using a focused laser beam to trap and manipulate small dielectric beads in 3D

Question 6

How does it work?

Answer 6

The bead is drawn toward the laser focus due to the gradient force of light; displacement generates a measurable restoring force

F = K / d
F = force (pN)
k = trap stiffness (pN/nm)
d = displacement (nm)

Question 7

What is the typical precision of optical tweezers?

Answer 7

Forces of a few picoNewtons and nanometer-scale displacements

Question 8

What is the light source typically used?

Answer 8

Infrared YAG laser (non-damaging to biological samples)

Question 9

How can DNA be studied using optical tweezers?

Answer 9

DNA is tethered between a trapped bead and a surface; laser trap applies stretching or tension → measures DNA–protein interactions

Question 10

Which biological systems are commonly studied?

Answer 10

Motor proteins (e.g., myosin, kinesin, dynein)
DNA-processing enzymes (helicases, polymerases)

Question 11

Why are molecular motors ideal for single-molecule studies?

Answer 11

Their movements (steps) are small (nm scale) and force generation (pN range) fits the detection capabilities of optical tweezers

Question 12

What does optical trapping reveal about motor proteins?

Answer 12

Step size, processivity, and force generation

Question 13

What is the energy source for these movements?

Answer 13

ATP hydrolysis → mechanical work

Question 14

What principle do magnetic tweezers use?

Answer 14

Apply magnetic forces and torques on paramagnetic beads attached to biomolecules (e.g., DNA)

Question 15

What can magnetic tweezers measure?

Answer 15

Stretching (extension changes)
Rotation/twisting (supercoiling)

Question 16

How is force controlled?

Answer 16

By moving external magnets closer/further from the sample

Question 17

Example application of magnetic tweezers?

Answer 17

Studying DNA supercoiling by topoisomerases or loop formation by DNA-binding proteins

Question 18

Typical force range of magnetic tweezers?

Answer 18

0.1–50 picoNewtons

Question 19

What are quantum dots (QDots)?

Answer 19

Bright, stable semiconductor nanocrystals that fluoresce at defined wavelengths

Question 20

Why are QDots useful for single-molecule tracking?

Answer 20

High brightness
Minimal photobleaching
Tunable emission colors
Compatible with live-cell imaging

Question 21

What do QDots allow researchers to visualize?

Answer 21

The movement and interactions of individual biomolecules in real time

Question 22

What is an example of QDots visualisation?

Answer 22

Tracking Myosin V movement — each head labeled with a different QDot (red and green) → alternating signals show hand-over-hand stepping (Warshaw et al., 2005)

Question 23

What are the typical step sizes observed?

Answer 23

Myosin V steps = 36 nm, corresponding to actin filament spacing

Question 24

How is the coordination between motor heads proven?

Answer 24

Alternating QDot signals show that one head releases and the other binds in turn

Question 25

What is the benefit of single-molecule fluorescence over bulk assays?

Answer 25

Detects asynchronous or rare events that ensemble averaging would hide

Question 26

Why are mechanical forces important biologically?

Answer 26

They regulate processes such as: DNA replication and repair Vesicle transport Muscle contraction Cell adhesion

Question 27

What are typical force ranges in biology?

Answer 27

1–100 picoNewtons (well within optical/magnetic tweezers sensitivity)