Table 1. Single-molecule techniques applied in studying helicase-mediated RNA folding and their technical specifications.
| Optical tweezers69,94 | AFM69,90 | smFRET70,175 | PIFE131,136 | |
|---|---|---|---|---|
| Spatial resolution |
0.1–2 nm |
0.1 nm in z plane 5 nm in x-y plane |
3–8 nm, depending on R0 |
below 4 nm |
| Temporal resolution |
10−4 s |
10−5 s |
Confocal: ≥ 5 × 10−5 s TIRFM: ≥ 10−3 s |
TIRFM: ≥ 10−3 s |
| Force range |
0.1–100 pN |
10–104 pN |
- |
- |
| Typical applications |
3D manipulation tethered assays Interaction assays |
high-force pulling and interaction assays |
protein and nucleic acid folding interaction assays, enzymology |
docking/undocking dynamics, protein displacement along RNA |
| Features |
low-noise and low-drift dumbbell geometry no labeling required, in vivo measurements (in principle) possible |
high-resolution imaging, no labeling, measurements under near-physiological conditions |
confocal, observe one molecule at a time; TIRFM, image several hundreds of single molecules simultaneously |
Image > 100 molecules at a time, rather robust against photo-physics |
| Limitations | photodamage, sample heating, nonspecific | large high-stiffness probe, large minimal force, nonspecific, surface technique | labeling required, photobleaching, dye-sample interaction | labeling, photo-bleaching, dye-sample interaction |