James T. Inman

Nanophotonic trapping for precise manipulation of biomolecular arrays

Optical trapping is a powerful manipulation and measurement technique widely employed in the biological and materials sciences1–8. Miniaturizing optical trap instruments onto optofluidic platforms holds promise for high throughput lab-on-chip applications9–16. However, a persistent challenge with existing optofluidic devices has been controlled and precise manipulation of trapped particles. Here we report a new class of on-chip optical trapping devices. Using photonic interference functionalities, an array of stable, three-dimensional on-chip optical traps is formed at the antinodes of a standing-wave evanescent field on a nanophotonic waveguide. By employing the thermo-optic effect via integrated electric microheaters, the traps can be repositioned at high speed (~ 30 kHz) with nanometer precision. We demonstrate sorting and manipulation of individual DNA molecules. In conjunction with laminar flows and fluorescence, we also show precise control of the chemical environment of a sample with simultaneous monitoring. Such a controllable trapping device has the potential for high-throughput precision measurements on chip.

Torque Measurement at the Single-Molecule Level

Methods for exerting and measuring forces on single molecules have revolutionized the study of the physics of biology. However, it is often the case that biological processes involve rotation or torque generation, and these parameters have been more difficult to access experimentally. Recent advances in the single-molecule field have led to the development of techniques that add the capability of torque measurement. By combining force, displacement, torque, and rotational data, a more comprehensive description of the mechanics of a biomolecule can be achieved. In this review, we highlight a number of biological processes for which torque plays a key mechanical role. We describe the various techniques that have been developed to directly probe the torque experienced by a single molecule, and detail a variety of measurements made to date using these new technologies. We conclude by discussing a number of open questions and propose systems of study that would be well suited for analysis with torsional measurement techniques.

Electro-optofluidics: achieving dynamic control on-chip

A vital element in integrated optofluidics is dynamic tuning and precise control of photonic devices, especially when employing electronic techniques which are challenging to utilize in an aqueous environment. We overcome this challenge by introducing a new platform in which the photonic device is controlled using electro-optical phase tuning. The phase tuning is generated by the thermo-optic effect using an on-chip electric microheater located outside the fluidic channel, and is transmitted to the optofluidic device through optical waveguides. The microheater is compact, high-speed (> 18 kHz), and consumes low power (~mW). We demonstrate dynamic optical trapping control of nanoparticles by an optofluidic resonator. This novel electro-optofluidic platform allows the realization of high throughput optofluidic devices with switching, tuning, and reconfiguration capability, and promises new directions in optofluidics.

DNA Y Structure: A Versatile, Multidimensional Single Molecule Assay

Optical trapping is a powerful single molecule technique used to study dynamic biomolecular events, especially those involving DNA and DNA-binding proteins. Current implementations usually involve only one of stretching, unzipping, or twisting DNA along one dimension. To expand the capabilities of optical trapping for more complex measurements would require a multidimensional technique that combines all of these manipulations in a single experiment. Here, we report the development and utilization of such a novel optical trapping assay based on a three-branch DNA construct, termed a “Y structure”. This multidimensional assay allows precise, real-time tracking of multiple configurational changes. When the Y structure template is unzipped under both force and torque, the force and extension of all three branches can be determined simultaneously. Moreover, the assay is readily compatible with fluorescence, as demonstrated by unzipping through a fluorescently labeled, paused transcription complex. This novel assay thus allows for the visualization and precision mapping of complex interactions of biomechanical events.

Electro-Optofluidicis: Achieving Dynamic Control On-Chip

Integrated optofluidics holds abundant promise for high throughput detection, study, and analysis of biochemical molecules and nanoparticles on chip. A vital element in integrated optofluidics is dynamic tuning and precise control of photonic devices, especially when employing electronic techniques which are challenging to utilize in an aqueous environment. We overcome this challenge by introducing a new platform in which the photonic device is controlled using electro-optical phase tuning. The phase tuning is generated by the thermo-optic effect using an on-chip electric microheater located outside the fluidic channel, and is transmitted to the optofluidic device through optical waveguides. The microheater is compact, high-speed (> 18 kHz), and consumes low power (∼ mW). We demonstrate dynamic optical trapping control of nanoparticles by an optofluidic resonator. This novel electro-optofluidic platform allows the realization of high throughput optofluidic devices with switching, tuning, and reconfiguration capability, and promises new directions in optofluidics.

Helicase promotes replication re-initiation from an RNA transcript

To ensure accurate DNA replication, a replisome must effectively overcome numerous obstacles on its DNA substrate. After encountering an obstacle, a progressing replisome often aborts DNA synthesis but continues to unwind. However, little is known about how DNA synthesis is resumed downstream of an obstacle. Here, we examine the consequences of a non-replicating replisome collision with a co-directional RNA polymerase (RNAP). Using single-molecule and ensemble methods, we find that T7 helicase interacts strongly with a non-replicating T7 DNA polymerase (DNAP) at a replication fork. As the helicase advances, the associated DNAP also moves forward. The presence of the DNAP increases both helicase’s processivity and unwinding rate. We show that such a DNAP, together with its helicase, is indeed able to actively disrupt a stalled transcription elongation complex, and then initiates replication using the RNA transcript as a primer. These observations exhibit T7 helicase’s novel role in replication re-initiation.During DNA replication, replicative helicases play an essential role for DNA unwinding to occur. Here the authors find that bacteriophage T7 helicase is also involved in replication re-initiation by interacting with a non-replicating DNAP and increasing unwinding rate.

Tunable nanophotonic array traps with enhanced force and stability

A nanophotonic trapping platform based on on-chip tunable optical interference allows parallel processing of biomolecules and holds promise to make single molecule manipulation and precision measurements more easily and broadly available. The nanophotonic standing wave array trap (nSWAT) device [Nat. Nanotechnol. 9, 448 (2014); Nano Lett. 16, 6661 (2016)] represents such a platform and can trap a large array of beads by the evanescent field of the standing wave of a nanophotonic waveguide and reposition them using an integrated microheater. In this paper, by taking a systematic design approach, we present a new generation of nSWAT devices with significant enhancement of the optical trapping force, stiffness, and stability, while the quality of the standing wave trap is resistant to fabrication imperfections. The device is implemented on a silicon nitride photonic platform and operates at 1064 nm wavelength which permits low optical absorption by the aqueous solution. Such performance improvements open a broader range of applications based on these on-chip optical traps.