Engineered ''aptazymes'' fuse in vitro selected aptamers with ribozymes to create allosteric enzymes as biosensing components and artificial gene regulatory switches through ligand-induced conformational rearrangement and activation. By contrast, activating ligand is employed as an enzymatic cofactor in the only known natural aptazyme, the glmS ribozyme, which is devoid of any detectable conformational rearrangements. To better understand this difference in biosensing strategy, we monitored by single molecule fluorescence resonance energy transfer (FRET) and 2-aminopurine (AP) fluorescence the global conformational dynamics and local base (un)stacking, respectively, of a prototypical drug-sensing aptazyme, built from a theophylline aptamer and the hammerhead ribozyme. Single molecule FRET reveals that a catalytically active state with distal Stems I and III of the hammerhead ribozyme is accessed both in the theophylline-bound and, if less frequently, in the ligand-free state. The resultant residual activity (leakage) in the absence of theophylline contributes to a limited dynamic range of the aptazyme. In addition, site-specific AP labeling shows that rapid local theophylline binding to the aptamer domain leads to only slow allosteric signal transduction into the ribozyme core. Our findings allow us to rationalize the suboptimal biosensing performance of the engineered compared to the natural aptazyme and to suggest improvement strategies. Our single molecule FRET approach also monitors in real time the previously elusive equilibrium docking dynamics of the hammerhead ribozyme between several inactive conformations and the active, long-lived, Y-shaped conformer.

Since the development of DNA origami by Paul Rothemund in 2006, the field of structural DNA nanotechnology has undergone tremendous growth. Through DNA origami and related approaches, self-assembly of specified DNA sequences allows for the ‘bottom-up’ construction of diverse nanostructures. By utilizing different sets of small ‘staple’ DNA strands to direct the folding of a long scaffold strand in diverse ways, DNA origami has particularly been incorporated into a variety of prototypical applications beyond the two-dimensional (2D) smiley face. In this review, the basis of DNA nanotechnology, methods of self-assembly, and Rothemund’s DNA origami breakthrough are discussed first. Next, some of the most promising applications of structural DNA nanotechnology since 2006 are summarized. These include utilizing DNA origami as a tool for creating 3D nanostructures (including DNA bricks), as well as structural (ligand capsid binding, viral capsid binding, DNA NanoOctahedron, DNA mold, photonic devices, energy transfer units), and dynamic (DNA box-with-lid, DNA nano-robot, DNA barges, amphipathic DNA structures, DNA nanocircuits) applications of DNA origami.

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.

Fluorescent nucleotide analogues, such as 2-aminopurine (2AP) and pyrrolo-C (PyC), have been extensively used to study nucleic acid local conformational dynamics in bulk experiments. Here we present a proof-of-principle approach using 2AP and PyC fluorescence at the single-molecule level. Our data show that ssDNA, dsDNA, or RNA containing both 2AP and PyC can be monitored using single-molecule fluorescence and a click chemistry immobilization method. We demonstrate that this approach can be used to monitor DNA and RNA in real time. This is the first reported assay using fluorescent nucleotide analogs at the single-molecule level. We anticipate that single 2AP or PyC fluorescence will have numerous applications in studies of DNA and RNA, including protein-induced base-flipping dynamics in protein–nucleic acid complexes.

Optical tweezers have been a fixture of microscopic cell manipulation since the 1990s. Arthur Ashkin’s seminal work has led to the advancement of optical tweezers as an effective tool for assay development in the fields of physics and nanotechnology. As an advanced application of cell manipulation, optical tweezers have facilitated the study of a multitude of cellular and molecular interactions within the greater field of nanotechnology. In the three decades since the optical tweezers’ rise to prominence, different and versatile assays have emerged that further explore the biochemical pathways integral for cell proliferation and communication. The most critical organelle implicated in the communication and protection of single cells includes the plasma membrane. In the past three decades, novel assays have emerged which examine the plasma membrane’s role in cell-to-cell interaction and the specific protein components that serve integral membrane functions for the cell as a whole. To further understand the extent to which optical tweezers have evolved as a critical tool for cellular membrane assessment within the field of nanotechnology, the various novel assays, including pulling, indentation, and stretching assays, will be reviewed in the current research sector.

A synthetic molecular nanorobot dubbed a "spider" composed of a streptavidin protein "body" bound to three biotinylated 8-17 deoxyribozyme-bearing "legs" completes the programmed task of traversing a predetermined DNA origami track. The track is assembled by folding and holding in place a long singlestranded DNA molecule into a two-dimensional landscape by using hundreds of short oligonucleotides ("staples") and hybridizing chimeric DNA-RNA substrates to specific staples to realize a pseudo-one-dimensional track. Spiders are designed to undergo a biased random walk as their legs repeatedly cleave oligonucleotide substrates on a track, causing the legs to more readily dissociate from the cleaved product and progress towards the uncleaved substrate. We image Cy3-labeled spiders and Cy5-labeled origami using total internal reflection fluorescence microscopy (TIRFM). By fitting Gaussian functions to the imaged point spread functions, we are able to monitor with nanometer spatial precision the real-time motion of the spiders along DNA origami tracks.

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