Many genome-processing reactions, such as transcription, replication and repair, generate DNA rotation. Methods that directly measure DNA rotation, including rotor bead tracking 1-3 , angular optical trap 4 , and magnetic tweezers 5 have helped unravel the action mechanisms of a range of genome-processing enzymes, such as RNA polymerase (RNAP) 6 , gyrase 2 , viral DNA packaging motor 7 , and DNA recombination enzymes 8 . However, despite the potential of rotation measurements to transform our understanding of genome-processing reactions, measuring DNA rotation remains a difficult task. The time resolution of existing methods is insufficient to track rotation induced by many enzymes under physiological conditions, and the measurement throughput is typically low. Here we introduce Origami-Rotor-Based Imaging and Tracking (ORBIT), a method that uses fluorescently labeled DNA origami rotors to track DNA rotation at the single-molecule level with millisecond time resolution. We used ORBIT to track DNA rotation resulted from unwinding by RecBCD, a helicase involved in DNA repair 9 , and transcription by RNAP. We characterized a series of events during RecBCD-induced DNA unwinding, including initiation, processive translocation, pausing and backtracking, and revealed an initiation mechanism that involves reversible, ATP-independent DNA unwinding and engagement of the RecB motor. During transcription by RNAP, we directly observed rotational steps corresponding to single-base-pair unwinding. We envision ORBIT will enable studies of a wide range of protein-DNA interactions.
We report the conversion between three crystalline polymorphs of a capped amino acid, N-acetyl-l-phenylalanyl-NH2, using mechanochemistry, with conversion between the α and γ polymorphs being reversible, depending on the milling conditions used. Solvent drop grinding of the α and β polymorphs with water yields the γ polymorph, whereas dry grinding of the β or γ polymorph yields the α polymorph. The α and β polymorphs are also accessible from solution (from methanol and water, respectively), and their structures were solved from single crystal diffraction data. The γ polymorph, so far only accessible mechanochemically, was solved and refined from powder X-ray diffraction data. The polymorphs show various degrees of crystallographic disorder, and the numbers of crystallographically independent molecules vary. These observations are supported by 13C and 15N magic angle spinning solid-state NMR data. Possible reasons for the formation of multiple polymorphs and their respective stability as a function of Z′, degree of disorder, and molecular shape and conformation are discussed. The results have implications for understanding the accessibility of new polymorphs of complex, low-symmetry organic solids with multiple dihedral degrees of freedom.
Porous crystalline dipeptides have gained recent attention for their potential as gas-storage materials. Within this large class is a group of dipeptides containing alanine, valine, and isoleucine with very similar crystal structures. We report the (13)C (carbonyl and Cα) and (15)N (amine and amide) solid-state NMR isotropic chemical shifts in a series of seven such isostructural porous dipeptides as well as shift tensor data for the carbonyl and amide sites. Using their known crystal structures and aided by ab initio quantum chemical calculations for the resonance assignments, we elucidate trends relating local structure, hydrogen-bonding patterns, and chemical shift. We find good correlation between the backbone dihedral angles and the Cα1 and Cα2 shifts. For the C1 shift tensor, the δ11 value shifts downfield as the hydrogen-bond distance increases, δ22 shifts upfield, and δ33 shows little variation. The C2 shift tensor shows no appreciable correlation with structural parameters. For the N2 tensor, δ11 shows little dependence on the hydrogen-bond length, whereas δ22 and δ33 both show a decrease in shielding as the hydrogen bond shortens. Our analysis teases apart some, but not all, structural contributors to the observed differences the solid-state NMR chemical shifts.
Highly sensitive detection of proteins is of central importance to biomolecular analysis and diagnostics. Conventional protein sensing assays, such as ELISAs, remain reliant on surface-immobilization of target molecules and multi-step washing protocols for the removal of unbound affinity reagents. These features constrain parameter space in assay design, resulting in fundamental limitations due to the underlying thermodynamics and kinetics of the immunoprobe-analyte interaction. Here, we present a new experimental approach for the quantitation of protein analytes through the implementation of an immunosensor assay that operates fully in solution and realises rapid removal of excess probe prior to detection without the need of washing steps. Our single-step optofluidic approach, termed digital immunosensor assay (DigitISA), is based on microfluidic electrophoretic separation combined with single-molecule laser-induced fluorescence microscopy and enables calibration-free in-solution protein detection and quantification within seconds. Crucially, the solution-based nature of our assay and the resultant possibility to use arbitrarily high probe concentrations combined with its fast operation timescale enables quantitative binding of analyte molecules regardless of the capture probe affinity, opening up the possibility to use relatively weak-binding affinity reagents such as aptamers. We establish and validate the DigitISA platform by probing a biomolecular biotin-streptavidin binding complex and demonstrate its applicability to biomedical analysis by quantifying IgE-aptamer binding. We further use DigitISA to detect the presence of a-synuclein fibrils, a biomarker for Parkinson's disease, using a low-affinity aptamer at high probe concentration. Taken together, DigitISA presents a fundamentally new route to surface-free specificity, increased sensitivity, and reduced complexity in state-of-the-art protein detection and biomedical analysis.
flies to human that it is implicated in multiple roles, including division of chromatin, gene activation, gene repression, and genome looping among others. CTCF contains 11 zinc fingers that can bind up to 35 bp of DNA including the well-conserved CCCTC sequence. Moreover, the binding sites orientation may play an important role in the regulation of enhancer-promoter choice, and the direction of looping formation. Here, we have used single molecule fluorescence resonance energy transfer (smFRET) to monitor and characterize CTCF-mediated DNA lopping. To this aim, we have designed a DNA substrate with a fluorophore-labelled (Cy3 and Cy5) binding site located at each end, such that loop formation brings together the two ends of the molecule thereby we will be able to observe FRET signal. Our data show that loop formation is only observed in the presence of two sequence specific binding sites and CTCF. Loop formation probability increases with protein concentration, yielding a dissociation constant of 10 nM and a Hill coefficient 1.6 in agreement with looping induced by dimers. Loop formation probability depends strongly on the distance between the binding sites, similar to previously reported models for the bacterial Lac operator. Contrary to what is observed in cells, our data show that CTCF does not favor any particular binding site orientation in vitro. Our results indicate that the preferred looping orientation observed in vivo likely results from additional regulatory looping factors.
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