Quantitative Characterization of Partitioning in Selection of DNA Aptamers for Protein Targets by Capillary Electrophoresis

Partitioning of protein–DNA complexes from protein-unbound DNA is a key step in selection of DNA aptamers. Conceptually, the partitioning step is characterized by two parameters: transmittance for protein-bound DNA (binders) and transmittance for unbound DNA (nonbinders). Here we present the first study to reveal how these transmittances depend on experimental conditions; such studies are pivotal to the effective planning and control of selection. Our focus was capillary electrophoresis (CE) which is a partitioning approach of high efficiency. By combining a theoretical model and experimental data, we evaluated the dependence of transmittances of binders and nonbinders on the molecular weight of protein target in two modes of CE-based partitioning: nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) and ideal-filter capillary electrophoresis (IFCE). Our data suggest that as the molecular weight of the protein target decreases: (i) the transmittance for binders remains close to unity in NECEEM but decreases drastically in IFCE and (ii) the transmittance for nonbinders increases orders of magnitude in NECEEM but remains relatively stable at a very low level in IFCE. To determine the optimal CE conditions for a given size of protein target, a balance between transmittances of binders and nonbinder must be reached; such a balance would ensure the collection of binders of sufficient purity and quantity. We conclude that, as a rule of thumb, IFCE is preferable for large-size protein targets while NECEEM should be the method of choice for small-size protein targets

Berberrubine Phosphate: A Selective Fluorescent Probe for Quadruplex DNA

A phosphate-substituted, zwitterionic berberine derivative was synthesized and its binding properties with duplex DNA and G4-DNA were studied using photometric, fluorimetric and polarimetric titrations and thermal DNA denaturation experiments. The ligand binds with high affinity toward both DNA forms (Kb = 2–7 × 105 M−1) and induces a slight stabilization of G4-DNA toward thermally induced unfolding, mostly pronounced for the telomeric quadruplex 22AG. The ligand likely binds by aggregation and intercalation with ct DNA and by terminal stacking with G4-DNA. Thus, this compound represents one of the rare examples of phosphate-substituted DNA binders. In an aqueous solution, the title compound has a very weak fluorescence intensity (Φfl < 0.01) that increases significantly upon binding to G4-DNA (Φfl = 0.01). In contrast, the association with duplex DNA was not accompanied by such a strong fluorescence light-up effect (Φfl < 0.01). These different fluorimetric responses upon binding to particular DNA forms are proposed to be caused by the different binding modes and may be used for the selective fluorimetric detection of G4-DNA.

SPACE exploration of chromatin proteome to reveal associated RNA-binding proteins

Chromatin is composed of many proteins that mediate intermolecular transactions with the genome. Comprehensive knowledge of these components and their interactions is necessary for insights into gene regulation and other activities; however, reliable identification of chromatin-associated proteins remains technically challenging. Here, we present SPACE (Silica Particle Assisted Chromatin Enrichment), a stringent and straightforward chromatin-purification method that helps identify direct DNA-binders separately from chromatin-associated proteins. We demonstrate SPACE’s unique strengths in three experimental set-ups: the sensitivity to detect novel chromatin-associated proteins, the quantitative nature to measure dynamic protein use across distinct cellular conditions, and the ability to handle 10-25 times less starting material than competing methods. In doing so, we reveal an unforeseen scale of association between over 500 nuclear RNA-binding proteins (RBPs) with chromatin and DNA, providing new insights into their roles as important regulators of genome maintenance and chromatin composition. Applied to iPSC-derived neural precursors, we discover a new role for the amyotrophic lateral sclerosis (ALS)-causing Valosin Containing Protein (VCP) in recruiting DNA-damage components to chromatin, thus paving the way for molecular mechanistic insights into the disease. SPACE is a fast and versatile technique with many applications.

Recent Progress in Polynuclear Ruthenium Complex-Based DNA Binders/Structural Probes and Anticancer Agents

Ruthenium complexes have stood out by several mononuclear complexes which have entered into clinical trials, such as imidazolium [trans-RuCl4(1H-imidazole)(DMSO-S)] (NAMI-A) and ([Ru(II)(4,4&#039;-dimethyl-2,2&#039;-bipyridine)2-(2(2&#039;-,2&#039;&#039;:5&#039;&#039;,2&#039;&#039;&#039;-terthiophene)-imidazo[4,5-f] [1,10]phenanthroline)] 2+) (TLD-1433), opening a new avenue for developing promising ruthenium-based anticancer drugs alternative to Cisplatin. Polynuclear ruthenium complexes were reported to exhibit synergistic and/or complementary effects: the enhanced DNA structural recognition and DNA binding as well as in vitro anticancer activities. This review overviews some representative polynuclear ruthenium complexes acting as DNA structural probes, DNA binders and in vitro anticancer agents, which were developed during last decades. These complexes are reviewed according to two main categories of homo-polynuclear and hetero-polynuclear complexes, each of which is further clarified into the metal centers linked by rigid and flexible bridging ligands. The perspective, challenges and future efforts for investigations into these exciting complexes are pointed out or suggested.