230 6.4  Magnetic Force Methods

However, a particular advantage of magnetic tweezers is that their relatively easy ability to

rotate a particle, in the form of a magnetic bead, compared to technically less trivial optical

rotation methods, which enables controllable torque to be applied to a single biomolecule

tethered to bead provided appropriate torsional constraints that are inserted into the links

between the tether and slide and tether and bead (in practice these are just multiple repeats

of the chemical conjugation groups at either end of the biomolecule). This is a more direct

and technically simpler method than can be achieved for optical tweezers, which would need

either to utilize an extended optical handle or use the rotating polarization of a non-​Gaussian

mode laser.

Magnetic tweezers–​mediated torque control has been used on DNA–​protein complexes,

for example, to study DNA replication. DNA in living cells is normally a negative supercoiled

structure (see Chapter 2), with the supercoiling moderated by topoisomerase enzymes.

However, to undergo replication or repair, or to express peptides and proteins from the

genes, this supercoiled structure needs first to relax into an uncoiled conformation. To access

the individual strands of the double helix then requires this helical structure itself to be

unwound, which in turn is made possible by enzymes called helicases. It is likely that many of

these torque-​generating molecular machines work in a highly coordinated fashion.

A disadvantage of magnetic tweezers over optical tweezers is that they are slower by a

factor of ~10³ since they do not utilize fast AOD components as optical tweezers can and

traditionally require using relatively large micron-​sized beads to have a sufficiently large mag­

netic moment but with the caveat of a relatively large frictional drag, which ultimately limits

how fast they can respond to changes in external B-​field—​a typical bandwidth for magnetic

tweezers is ~1 kHz, so they are limited to detect changes over time scales >1 ms. Also, trad­

itionally, it has not been possible to visualize a molecule that has been stretched through

application of magnetic tweezers at the same time as monitoring its extension and force, for

example, using fluorescence microscopy if the biomolecule in question can be tagged with

a suitable dye. This is because the geometry of conventional magnetic tweezers is such that

the stretched molecule is aligned parallel to the optic axis of the microscope and so cannot

be visualized extended in the lateral focal plane. To solve this problem, some groups are

developing transverse magnetic tweezers systems (Figure 6.5b). The main technical issue with

doing so is that there is often very confined space in the microscope stage region around a

sample to physically position magnets or coils in the same lateral plane as the microscope

slide. One way around this problem is to use very small electromagnetic coils, potentially

microfabricated, integrated into a bespoke flow cell.

Other recent improvements have involved using magnetic probes with a much higher

magnetic moment, which may allow for reductions in the size of the probe, thus incurring

less viscous drag, with consequent improvements to maximum sampling speeds. One

such probe uses a permalloy of nickel and chromium manufactured into a disk as small as

~100 nm diameter (Kim et al., 2009) and still have a sufficiently high magnetic moment to

KEY BIOLOGICAL

APPLICATIONS:

MAGNETIC FORCE TOOLS

Quantifying biological torque;

Molecular and cellular separation

and identification; Measuring

biomolecular mechanics.

FIGURE 6.5  Magnetic tweezers. Single-​molecule mechanical experiments can be performed

in both (a) vertical and (b) transverse geometries, for example, to probe the mechanical prop­

erties of DNA molecules and of machines, such as FtsK shown here, which translocate on DNA.