Gene machines

Abstract:

DNA nanotechnology makes use of the exquisite self-recognition of DNA in order to build on a molecular scale. Although static structures may find applications in structural biology and computer science, many applications in nanomedicine and nanorobotics require the additional capacity for controlled three-dimensional movement. DNA architectures can span three dimensions and DNA devices are capable of movement, but active control of well-defined three-dimensional structures has not been achieved. We demonstrate the operation of reconfigurable DNA tetrahedra whose shapes change precisely and reversibly in response to specific molecular signals. Shape changes are confirmed by gel electrophoresis and by bulk and single-molecule Förster resonance energy transfer measurements. DNA tetrahedra are natural building blocks for three-dimensional construction; they may be synthesized rapidly with high yield of a single stereoisomer, and their triangulated architecture conveys structural stability. The introduction of shape-changing structural modules opens new avenues for the manipulation of matter on the nanometre scale.

Abstract:

NT (neurotensin) is an endogenous tridecapeptide neurotransmitter found in the central nervous system and gastrointestinal tract. One receptor for NT, NTS1, belongs to the GPCR (G-protein-coupled receptor) superfamily, has seven putative transmembrane domains, and is being studied by a range of single-molecule, functional and structural approaches. To enable biophysical characterization, sufficient quantities of the receptor need to be expressed and purified in an active form. To this end, rat NTS1 has been expressed in Escherichia coli in an active ligand-binding form at the cell membrane and purified in sufficient amounts for structural biology studies either with or without fluorescent protein [YFP (yellow fluorescent protein) and CFP (cyan fluorescent protein)] fusions. Ligand binding has been demonstrated in a novel SPR (surface plasmon resonance) approach, as well as by conventional radioligand binding measurements. These improvements in production of NTS1 now open up the possibility of direct structural studies, such as solid-state NMR to interrogate the NT-binding site, EM (electron microscopy), and X-ray crystallography and NMR.

Abstract:

Alternating-laser excitation (ALEX) spectroscopy has recently been added to the single-molecule spectroscopy toolkit. ALEX monitors interaction and stoichiometry of biomolecules, reports on biomolecular structure by measuring accurate Förster resonance energy transfer (FRET) efficiencies, and allows sorting of subpopulations on the basis of stoichiometry and FRET. Here, we demonstrate that a simple combination of one continuous-wave donor-excitation laser and one directly modulated acceptor-excitation laser (Periodic Acceptor eXcitation) is sufficient to recapitulate the capabilities of ALEX while minimizing the cost and complexity associated with use of modulation techniques.

Abstract:

We introduce three-color alternating-laser excitation (3c-ALEX), a fluorescence resonance energy transfer (FRET) method that measures up to three intramolecular distances and complex interaction stoichiometries of single molecules in solution. This tool extends substantially the capabilities of two-color ALEX, which employs two alternating lasers to study molecular interactions (through probe stoichiometry S) and intramolecular distances (through FRET efficiency E), and sorts fluorescent molecules in multi-dimensional probe-stoichiometry and FRET-efficiency histograms. Probe-stoichiometry histograms allowed analytical sorting, identification, and selection of diffusing species; selected molecules were subsequently represented in FRET-efficiency histograms, generating up to three intramolecular distances. Using triply labeled DNAs, we established that 3c-ALEX enables 1), FRET-independent analysis of three-component interactions; 2), observation and sorting of singly, doubly, and triply labeled molecules simultaneously present in solution; 3), measurements of three intramolecular distances within single molecules from a single measurement; and 4), dissection of conformational heterogeneity with improved resolution compared to conventional single-molecule FRET. We also used 3c-ALEX to study large biomolecules such as RNA polymerase-DNA transcription complexes, and monitor the downstream translocation of RNA polymerase on DNA from two perspectives within the complex. This study paves the way for advanced single-molecule analysis of complex mixtures and biomolecular machinery.