Binu Kundukad (SMART), Piewen Cong (NUS), Johan R. C. van der Maarel (NUS), and Patrick S. Doyle (MIT/SMART)

HU is a protein that plays a role in various bacterial processes including compaction, transcription and replication of the genome. Here, we use atomic force microscopy to study the effect of HU on the stiffness and supercoiling of double-stranded DNA. First, we measured the persistence length, height profile, contour length and bending angle distribution of the DNA–HU complex after different incubation times of HU with linear DNA. We found that the persistence and contour length depend on the incubation time. At high concentrations of HU, DNA molecules first become stiff with a larger value of the persistence length. The persistence length then decreases over time and the molecules regain the flexibility of bare DNA after 2 h. Concurrently, the contour length shows a slight increase. Second, we measured the change in topology of closed circular relaxed DNA following binding of HU. Here, we observed that HU induces supercoiling over a similar time span as the measured change in persistence length. Our observations can be rationalized in terms of the formation of a nucleoprotein filament followed by a structural rearrangement of the bound HU on DNA. The rearrangement results in a change in topology, an increase in bending flexibility and an increase in contour length through a decrease in helical pitch of the duplex.

Representative AFM image, corresponding contour and contour length distribution for bare DNA and incubated with 900nM HU for 15 min, 1 h and 2 h from top to bottom, respectively. The scale bars denote 100 nm. The distributions are obtained from a pool of ~40 molecules each.

Yun Soo Kim (SMART), Binu Kundukad (SMART) Abdollah Allahverdi (NTU),  Lars Nordenskold (NTU), Patrick S. Doyle (MIT/SMART) and Johan R. C. van der Maarel (NUS)

Topoisomerase II (TOP2) regulates the topology of DNA by catalysis of a double strand passage reaction. Inhibition of this reaction prevents cell replication, and, thus, is a pathway targeted by anticancer drugs. Some details regarding the cell-killing mechanism are unknown and assays to screen for anticancer drugs are not well established. Here, we have studied the gelation of linear and circular DNA using microrheology assays.

	Circular DNA (Cosmid) forms a gel when incubated with AMPPNP and ICRF where as  linear DNA (lambda) does not form a gel under similar conditions

 

Adam S. Zeiger (MIT/SMART), Felicia C. Loe (SMART), Ran Li (MIT), Michael Raghunath (NUS), and Krystyn J. Van Vliet (MIT/SMART)

A crucial part of advanced in vitro platform design is increasing fidelity to physiological environments when necessary. For example, contemporary in vitro culture systems lack the level of macromolecular concentrations that is found in vivo, and thus poorly represent those conditions. We have induced macromolecular crowding in vitro using synthetic macromolecular globules of nm-scale radius at in vivo levels of fractional volume occupancy. We quantified the impact of induced crowding on the extracellular and intracellular protein organization of human mesenchymal stem cells (MSCs) via immunocytochemistry, atomic force microscopy (AFM), and AFM-enabled nano-indentation. Macromolecular crowding in extracellular culture media is found to directly induce supramolecular assembly and alignment of extracellular matrix proteins deposited by cells, which in turn increases alignment of the actin cytoskeleton. The resulting cell-matrix reciprocity further affects adhesion, proliferation, and migration behaviour of MSCs. Macromolecular crowding can thus aid the design of more physiologically relevant in vitro studies and devices for MSCs and other cells, by increasing the fidelity between materials synthesized by cells in vivo and in vitro. Thus, this project within Thrust 1 has immediate implications in Thrust 2, Multifunctional Cytometry.

Dai Liang (SMART), Jeremy Jones (MIT), Benjamin Renner (MIT), Patrick Doyle (MIT/SMART), Jie Yan (NUS) and Johan van der Maarel (NUS)

We have studied conformation and dynamics of DNA in nanofluidic devices. The confinement can be applied to stretch DNA, modify DNA topology, slow down DNA dynamics, and alter the responses to external fields such as flow and electric fields.  The major methodologies are Monte-Carlo simulation and scaling theory. The results provide new insights to the transition regime from weak to strong confinement. We find that the confinement effect on knotting probability can be monotonic or non-monotonic, depending on the characteristic lengths, including DNA contour length, persistence length and the effective chain width (ionic strength dependent). In addition to well-known similarity in classic de Gennes/Pincus regime, we reveal the subtle difference in the extended de Gennes/Pincus regime. Overall, our research deepens the understanding of DNA behaviors in confinement, especially in the transition regime (channel width ~ 100 nm), which should benefit the practical applications of micro-/nano- fluidic devices to DNA as well as other biopolymers.

Ning Du (SMART), Jing Tang (MIT), Jeremy J. Jones (MIT), Ben Renner (MIT), Patrick Doyle (MIT/SMART), Jie Yan (NUS) and Johan van der Maarel (NUS)

Electric fields have been widely used to transport and manipulate DNA in lab-on-a-chip devices with applications in molecular genetics, nucleic acid-based diagnostics, and fundamental studies of polyelectrolytes. We have experimentally demonstrated that addition of small charge-neutral flexible polymers to an ionic buffer can promote compression of single DNA under a uniform electric field. This phenomenon lies in stark contrast with widely observed extension of DNA during capillary electrophoresis in dilute solutions of larger charge-neutral polymers. We propose that this dissimilarity arises from differences in the magnitude of tension created by DNA-polymer collisions, controlled by the properties of the solute polymer. For solutions of small (Mw ≤ 410k) dextran, we show that their ability to promote the compression of single DNA is consistent with a mechanism proposed for aggregation in concentrated macro-ion dispersions by showing collapse of the experimental data. With increasing importance of micro/nanofluidic devices in single molecule research, it is necessary to effectively manipulate DNA conformations. By tuning the solvent of DNA via addition of polymers of varying size and rigidity, one can responsively control DNA configurations via the application of small to moderate electric fields. These insights shed light on the fundamental behavior of polyelectrolytes and provide guidelines for working with the electro-kinetically controlled micro/nanofluidic systems.

Schematic of DNA electrophoresing in dilute solutions of polymers. Top: DNA at rest prior to application of electric field. Bottom: Steady state configurations long after switching on the electric field. (A) DNA with “small” polymers (B) DNA with “large” polymers Polymers entrained with DNA contour is shown in red.

Siew-Kit Hoi (SMART), Taishi Zhang (NUS), Zhenping Guan (NUS), Matt Lang (MIT/SMART), Patrick Doyle (MIT/SMART), Yan Jie (NUS) and Qing-Hua Xu (NUS)

We have developed an integrated fluorescence and force (through: optical, fluidic, magnetic) microscope appropriate for single molecule and cell studies. Custom machining and general assembly of microscope were completed at MIT, and the entire system transferred and commissioned at BioSyM SMART' opto-mechanical suite in CREATE. The instrumentaion features double trap geometry. The combination of the optical tweezers microscopy and single molecule fluorescence spectroscopy has provided the capability for simultaneous manipulation and monitoring of molecular structure in real time. The optical traps can be employed to supply controlled external loads while fluorescent molecules report the concurrent information about macromolecular structure.

Multispectral force-fluorescence microscope at SMART-BioSyM

(Bruce Tidor (MIT/SMART-BioSyM) and Sudipta Samanta (SMART-BioSyM)

At the molecular level, force induced conformational change can alter protein activity; one possible mechanism for altering activity is through changes in chemical properties such as binding affinity and enzyme activity. In this framework, force-induced alteration of binding affinity, as but one example, would depend on characteristics of the applied force, including direction, magnitude, and point of application.  Currently the property space of how applied force affects chemical properties like binding affinity is poorly understood due to the lack of effective experimental data. Here we develop a computational approach and analysis for gaining insight into force modulated binding properties.

The main goal in this project is to understand the molecular basis of mechanotransduction. For this we apply free energy simulation to study the binding response to applied force of focal adhesion targeting domain of the focal adhesion kinase bound to the paxillin motif. Applied force on the protein in different directions helps understand how its direction and magnitude can affect structure-function relationship in biologically relevant systems.

(Bruce Tidor (MIT/SMART-BioSyM), Pradeep Anand Ravindranath (NUS) and Nathaniel W.Silver (MIT)

The goal is to design mutation resistant inhibitors for hepatitis C virus (HCV) protease using structure-based computational modeling applying inverse design methods to implement the substrate envelope hypothesis. The protease structure was prepared for modeling and design studies using standard approaches. The other important residues that constituted the active substrate to be cleaved were also successfully modeled and substrate envelope is created. According to the synthesis requirement, the scaffold and building blocks are prepared for the design.

Mechanical characteristics of single biological cells are used to identify and possibly leverage interesting differences among cells or cell populations. Fluidity—hysteresivity normalized to the extremes of an elastic solid or a viscous liquid—can be extracted from, and compared among, multiple rheological measurements of cells: creep compliance vs. time, complex modulus vs. frequency, and phase lag vs. frequency. With multiple strategies available for acquisition of this nondimensional property, fluidity may serve as a useful and robust parameter for distinguishing cell populations, and for understanding the physical origins of deformability in soft matter. Here, for three disparate eukaryotic cell types deformed in the suspended state via optical stretching, we examine the dependence of fluidity on chemical and environmental influences around a time scale of 1 s. We find that fluidity estimates are consistent in the time and the frequency domains under a structural damping (power-law or fractional derivative) model, but not under an equivalent-complexity lumped component (spring-dashpot) model; the latter predicts spurious time constants. Although fluidity is suppressed by chemical crosslinking, we find that adenosine triphosphate (ATP) depletion in the cell does not measurably alter the parameter, and thus conclude that active ATP-driven events are not a crucial enabler of fluidity during linear viscoelastic deformation of a suspended cell. Finally, by using the capacity of optical stretching to produce near-instantaneous increases in cell temperature, we establish that fluidity increases with temperature—now measured in a fully suspended, sortable cell without the complicating factor of cell-substratum adhesion.

Fluidity is not detectably altered by extended in vitro culturing of primary hMSCs.