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Nucleic acid junctions are key to many biological functions from recombination and repair to viral nucleic acid insertion and are an attractive, functional biomolecular target. We describe a quadruple-stranded diplatinum helicate that binds both three-way (3WJ) and four-way DNA junctions (4WJ). This allows us to probe the relative importance of size and shape in junction-binder design. Despite the helicate's tetragonal symmetry/shape being compatible with the 4WJ, microscale thermophoresis (MST), isothermal calorimetry (ITC), and gel electrophoresis competition experiments demonstrate that this metallo-supramolecule displays a stronger affinity for 3WJs (K_d = 12 nM) than for 4WJs (K_d > 4 μM) and other DNA structures. The experimental findings are supported by molecular dynamics simulations that reveal the critical role of size. While the open form of the 4WJ is promoted when the helicate is in the cavity, the helicate's small size means it is unable to maintain π contacts with all four junction base-pairs simultaneously. Although the helicate is slightly too large for the smaller 3WJ cavity, simulations and experiments show that it can open up the cavity (increasing the junction's hydrodynamic radius) by disrupting a base pair. The flexible helicate also responds to the cavity upon binding by favoring one enantiomer and allowing the helicate to adopt a stable final structure inside the 3WJ that is an induced fit of the two dynamic structures (supramolecule and DNA). This contrasts with previous lock-and-key examples of junction recognition and opens up new possibilities for how to design DNA and RNA junction-binding compounds. Show less

Adjuvant chemotherapy after pulmonary metastasectomy for colorectal cancer may reduce recurrence and improve survival rates; however, the benefits of this treatment are limited by the significant side effects that accompany it. The development of a novel in vivo lung perfusion (IVLP) platform would permit the localized delivery of high doses of chemotherapeutic drugs to target residual micrometastatic disease. Nonetheless, it is critical to continuously monitor the levels of such drugs during IVLP administration, as lung injury can occur if tissue concentrations are not maintained within the therapeutic window. This paper presents a simple chemical-biopsy approach based on sampling with a small nitinol wire coated with a sorbent of biocompatible morphology and evaluates its applicability for the near-real-time in vivo determination of oxaliplatin (OxPt) in a 72-h porcine IVLP survival model. To this end, the pigs underwent a 3-h left lung IVLP with 3 doses of the tested drug (5, 7.5, and 40 mg/L), which were administered to the perfusion circuit reservoir as a bolus after a full perfusion flow had been established. Along with OxPt levels, the biocompatible solid-phase microextraction (SPME) probes were employed to profile other low-molecular-weight compounds to provide spatial and temporal information about the toxicity of chemotherapy or lung injury. The resultant measurements revealed a rather heterogeneous distribution of OxPt (over the course of IVLP) in the two sampled sections of the lung. In most cases, the OxPt concentration in the lung tissue peaked during the second hour of IVLP, with this trend being more evident in the upper section. In turn, OxPt in supernatant samples represented ∼25% of the entire drug after the first hour of perfusion, which may be attributable to the binding of OxPt to albumin, its sequestration into erythrocytes, or its rapid nonenzymatic biotransformation. Additionally, the Bio-SPME probes also facilitated the extraction of various endogenous molecules for the purpose of screening biochemical pathways affected during IVLP (i.e., lipid and amino acid metabolism, steroidogenesis, or purine metabolism). Overall, the results of this study demonstrate that the minimally invasive SPME-based sampling approach presented in this work can serve as (pre)clinical and precise bedside medical tool. Show less