A fluorescent reporter on electrostatic DNA-ligand interactions.

Among the various types of interactions between biomolecules, electrostatic interactions dominate as these are long-range interactions and are often a generic first step in the recruitment of specific ligands. DNA, being a highly charged molecule, attracts a plethora of molecules. Interactions between DNA and proteins or small molecules shape the overall function of the cell. Various processes such as DNA replication, DNA repair, synthesis of mRNA, and packaging of DNA are mediated by interactions between protein molecules and DNA that are predominantly electrostatic. Here, we present a fluorescence resonance energy transfer (FRET)-based probe which can report on the electrostatic interactions between the negatively-charged DNA and positively-charged metal ions, oligopeptides, as well as DNA groove-binding drug molecules. The simplicity, sensitivity, and versatility of the DNA-based probe makes it suited for applications where specific protein-DNA interactions can be probed, and DNA-binding drugs can be discovered in high-throughput screens of compound libraries. This is particularly relevant given that some of the most potent antitumor and antimicrobial drugs associate with DNA electrostatically.

Entropy and Entropic Forces to Model Biological Fluids.

Living cells are complex systems characterized by fluids crowded by hundreds of different elements, including, in particular, a high density of polymers. They are an excellent and challenging laboratory to study exotic emerging physical phenomena, where entropic forces emerge from the organization processes of many-body interactions. The competition between microscopic and entropic forces may generate complex behaviors, such as phase transitions, which living cells may use to accomplish their functions. In the era of big data, where biological information abounds, but general principles and precise understanding of the microscopic interactions is scarce, entropy methods may offer significant information. In this work, we developed a model where a complex thermodynamic equilibrium resulted from the competition between an effective electrostatic short-range interaction and the entropic forces emerging in a fluid crowded by different sized polymers. The target audience for this article are interdisciplinary researchers in complex systems, particularly in thermodynamics and biophysics modeling.

FRET-Based Probe for High-Throughput DNA Intercalator Drug Discovery and In Vivo Imaging.

Molecules that bind DNA by intercalating its bases remain among the most potent cancer therapies and antimicrobials due to their interference with DNA-processing proteins. To accelerate the discovery of novel intercalating drugs, we designed a fluorescence resonance energy transfer (FRET)-based probe that reports on DNA intercalation, allowing rapid and sensitive screening of chemical libraries in a high-throughput format. We demonstrate that the method correctly identifies known DNA intercalators in approved drug libraries and discover previously unreported intercalating compounds. When introduced in cells, the oligonucleotide-based probe rapidly distributes in the nucleus, allowing direct imaging of the dynamics of drug entry and its interaction with DNA in its native environment. This enabled us to directly correlate the potency of intercalators in killing cultured cancer cells with the ability of the drug to penetrate the cell membrane. The combined capability of the single probe to identify intercalators in vitro and follow their function in vivo can play a valuable role in accelerating the discovery of novel DNA-intercalating drugs or repurposing approved ones.

A Molecular Sensor Reveals Differences in Macromolecular Crowding between the Cytoplasm and Nucleoplasm.

We describe a molecular sensor that reports, using fluorescence resonance energy transfer (FRET), on the degree of macromolecular crowding in different cellular compartments. The oligonucleotide-based sensor is sensitive to changes in the volume fraction of macromolecules over a wide range in vitro and, when introduced in cells, rapidly distributes and shows a striking contrast between the cytosol and the nucleus. This contrast can be modulated by osmotic stress or by using a number of drugs that alter chromatin organization within the nucleus. These findings suggest that the sensor can be used as a tool to probe chromosome organization. Further, our finding that the cell maintains different degrees of macromolecular crowding in the cytoplasm and nucleoplasm has implications on molecular mechanisms since crowding can alter protein conformations, binding rates, reaction kinetics, and therefore protein function.

A microfluidic platform for on-demand formation and merging of microdroplets using electric control.

We discuss a microfluidic system in which (programmable) local electric fields originating from embedded and protected electrodes are used to control the formation and merging of droplets in a microchannel. The creation of droplets-on-demand (DOD) is implemented using the principle of electrowetting. Combined with hydrodynamic control, the droplet size and formation frequency can be varied independently. Using two synchronized DOD injectors, merging-on-demand (MOD) is achieved via electrocoalescence. The efficiency of MOD is 98% based on hundreds of observations. These two functionalities can be activated independently.

Force spectroscopy and fluorescence microscopy of dsDNA-YOYO-1 complexes: implications for the structure of dsDNA in the overstretching region.

When individual dsDNA molecules are stretched beyond their B-form contour length, they reveal a structural transition in which the molecule extends 1.7 times its contour length. The nature of this transition is still a subject of debate. In the first model, the DNA helix unwinds and combined with the tilting of the base pairs (which remain intact), results in a stretched form of DNA (also known as S-DNA). In the second model the base pairs break resulting effectively in two single-strands, which is referred to as force-induced melting. Here a combination of optical tweezers force spectroscopy with fluorescence microscopy was used to study the structure of dsDNA in the overstretching regime. When dsDNA was stretched in the presence of 10 nM YOYO-1 an initial increase in total fluorescence intensity of the dye-DNA complex was observed and at an extension where the dsDNA started to overstretch the fluorescence intensity leveled off and ultimately decreased when stretched further into the overstretching region. Simultaneous force spectroscopy and fluorescence polarization microscopy revealed that the orientation of dye molecules did not change significantly in the overstretching region (78.0 degrees +/- 3.2 degrees). These results presented here clearly suggest that, the structure of overstretched dsDNA can be explained accurately by force induced melting.