A Bird's Eye View

Abstract

Recent improvements in methods of single-particle fluorescence tracking have permitted detailed studies of molecular motion on the nanometer scale. In a quest to introduce these tools to the burgeoning field of DNA nanotechnology, we have exploited fluorescence imaging with one-nanometer accuracy (FIONA) and single-molecule high-resolution colocalization (SHREC) to monitor the diffusive behavior of synthetic molecular walkers, dubbed “spiders”, at the single-molecule level. Here we discuss the imaging methods used, results from tracking individual spiders on pseudo-one-dimensional surfaces, and some of the unique experimental challenges presented by the low velocities (~3 nm/min) of these nanowalkers. These experiments demonstrate the promise of fluorescent particle tracking as a tool for the detailed characterization of synthetic molecular nanosystems at the single-molecule level.

Kinetic fingerprinting to identify and count single nucleic acids

“…SiMREPS experiments were performed using either a previously described prism-type TIRF microscope 19 (HeLa extract experiments) or a Olympus IX-81 objective-type TIRF microscope equipped with a 60× oil-immersion objective (APON 60XOTIRFM, 1.49NA) as well as Cell^TIRF and z-drift control modules (all other experiments). For prism-type TIRF experiments, fluidic sample cells were constructed using two pieces of double-sided tape sandwiched between a quartz slide and glass coverslip as previously described 19 (Supplementary Fig.…”

“…For prism-type TIRF experiments, fluidic sample cells were constructed using two pieces of double-sided tape sandwiched between a quartz slide and glass coverslip as previously described 19 (Supplementary Fig. 10a).…”

Riboswitch Structure and Dynamics by smFRET Microscopy

“…16.2B) (Michelotti, de Silva, Johnson-Buck, Manzo & Walter, 2010; Roy et al, 2008; Suddala et al, 2013). To this end, the slide surface needs to be thoroughly cleaned using a multi-step protocol to remove any organic impurities (Krishnan et al, 2013; Zhao et al, 2009).…”

“…In the end, the slide surface is flamed thoroughly using a propane torch to destroy any remaining organic impurities. A microfluidic channel is made on the clean slides by sandwiching two strips of double-sided sticky tape ~4–6 mm apart between the quartz slide and a clean rectangular glass coverslip (Michelotti et al, 2010; Roy et al, 2008). The edges of the channel are sealed with epoxy (for example, Hardman DOUBLE/BUBBLE Fast-Setting Epoxy) to prevent leakage of the buffer.…”

Single-Molecule Pull-Down FRET to Dissect the Mechanisms of Biomolecular Machines

“…In a final step, the surface is reacted for 30 minutes with a solution of 20 mg/mL disulfosuccinimidyltartrate (sulfo-DST) in 1 M sodium bicarbonate to passivate any unreacted amino groups. Sample chambers are constructed by attaching a cover slip to the slide using double-sided tape and sealing the ends with epoxy glue, and assembling tubing to allow sample to be injected into holes pre-drilled into the slide (Michelotti, de Silva, Johnson-Buck, Manzo & Walter, 2010). As described above, the sample chamber is incubated with a solution of streptavidin immediately before use, allowing a biotinylated sample to be immobilized.…”