Development of DNA aptamers for visualization of glial brain tumors and detection of circulating tumor cells.

Here, we present DNA aptamers capable of specific binding to glial tumor cells in vitro, ex vivo, and in vivo for visualization diagnostics of central nervous system tumors. We selected the aptamers binding specifically to the postoperative human glial primary tumors and not to the healthy brain cells and meningioma, using a modified process of systematic evolution of ligands by exponential enrichment to cells; sequenced and analyzed ssDNA pools using bioinformatic tools and identified the best aptamers by their binding abilities; determined three-dimensional structures of lead aptamers (Gli-55 and Gli-233) with small-angle X-ray scattering and molecular modeling; isolated and identified molecular target proteins of the aptamers by mass spectrometry; the potential binding sites of Gli-233 to the target protein and the role of post-translational modifications were verified by molecular dynamics simulations. The anti-glioma aptamers Gli-233 and Gli-55 were used to detect circulating tumor cells in liquid biopsies. These aptamers were used for in situ, ex vivo tissue staining, histopathological analyses, and fluorescence-guided tumor and PET/CT tumor visualization in mice with xenotransplanted human astrocytoma. The aptamers did not show in vivo toxicity in the preclinical animal study. This study demonstrates the potential applications of aptamers for precise diagnostics and fluorescence-guided surgery of brain tumors.

First-principles characterization of the energy landscape and optical spectra of green fluorescent protein along the A→I→B proton transfer route.

Structures and optical spectra of the green fluorescent protein (GFP) forms along the proton transfer route A→I→B are characterized by first-principles calculations. We show that in the ground electronic state the structure representing the wild-type (wt) GFP with the neutral chromophore (A-form) is lowest in energy, whereas the systems with the anionic chromophore (B- and I-forms) are about 1 kcal/mol higher. In the S65T mutant, the structures with the anionic chromophore are significantly lower in energy than the systems with the neutral chromophore. The role of the nearby amino acid residues in the chromophore-containing pocket is re-examined. Calculations reveal that the structural differences between the I- and B-forms (the former has a slightly red-shifted absorption relative to the latter) are based not on the Thr203 orientation, but on the Glu222 position. In the case of wt-GFP, the hydrogen bond between the chromophore and the His148 residue stabilizes the structures with the deprotonated phenolic ring in the I- and B-forms. In the S65T mutant, concerted contributions from the His148 and Thr203 residues are responsible for a considerable energy gap between the lowest energy structure of the B type with the anionic chromophore from other structures.

On quantum mechanical--molecular mechanical (QM/MM) approaches to model hydrolysis of acetylcholine by acetylcholinesterase.

We re-visited the results of quantum mechanics--molecular mechanics (QM/MM) approaches aiming to construct the reaction energy profile for the acylation stage of acetylcholine hydrolysis by acetylcholinesterase. The main emphasis of this study was on the energy of the first tetrahedral intermediate (TI) relative to the level of the enzyme-substrate (ES) complex for which contradictory data from different works had been reported. A new series of stationary points on the potential energy surface was calculated by using electronically embedding QM/MM schemes when starting from the crystal structure mimicking features of the reaction intermediate (PDB ID: 2VJA). A thoughtful analysis allows us to conclude that the energy of TI should be lower than that of ES, and a proper treatment of contributions from the oxyanion hole residues accounts for their relative positions.

Toward Molecular-Level Characterization of Photoinduced Decarboxylation of the Green Fluorescent Protein: Accessibility of the Charge-Transfer States.

Irradiation of the green fluorescent protein (GFP) by intense violet or UV light leads to decarboxylation of the Glu222 side chain in the vicinity of the chromophore (Chro). This phenomenon is utilized in optical highlighters, such as photoactivatable GFP (PA-GFP). Using state-of-the-art quantum chemical calculations, we investigate the feasibility of the mechanism proposed in the experimental studies [van Thor et al. Nature Struct. Biol.2002, 9, 37-41; Bell et al. J. Am. Chem. Soc.2003, 125, 37-41]. It was hypothesized that a primary event of this photoconversion involves population of a charge-transfer (CT) state via either the first excited state S1 when using longer wavelength (404 and 476 nm) or a higher excited state when using higher energy radiation (254 and 280 nm). Based on the results of electronic structure calculations, we identify these critical CT states (produced by electron transfer from Glu to electronically excited Chro) and show that they are accessible via different routes, i.e., either directly, by one-photon absorption, or through a two-step excitation via S1. The calculations are performed for model systems representing the chromophore and the key nearby residues using two complementary approaches: (i) the multiconfigurational quasidegenerate perturbation theory of second order with the occupation restricted multiple active space scheme for configuration selection in the multiconfigurational self-consistent field reference; and (ii) the single-reference configuration interaction singles method with perturbative doubles that does not involve active space selection. We examined electronic transitions with nonzero oscillator strengths in the UV and visible range between the electronic states involving the Chro and Glu residues. Both methods predict the existence of CT states with nonzero oscillator strength in the UV range and a local excited state of the chromophore accessible via S1 that may lead to the target CT state. The results suggest several possible scenarios for the primary photoconversion event. We also demonstrate that the point mutation Thr203His exploited in PA-GFP results in shifting the light wavelength to access the CT up to 20 nm, which suggests a possibility of a rational design of photoactivatable proteins in silico.

Correlation between the substrate structure and the rate of acetylcholinesterase hydrolysis modeled with the combined quantum mechanical/molecular mechanical studies.

The combined quantum mechanical-molecular mechanical (QM/MM) based computational scheme for modeling the structure-reaction rate correlations was elaborated for the hydrolysis of the set of neutral esters in the active site of acetylcholinesterase (AChE). The energy barriers of hydrolysis were estimated on the basis of the equilibrium geometry configurations of the enzyme-substrate (ES) complexes. The obtained correlation between the rate of hydrolysis and the hydrophobicity of the substrate leaving group is consistent with experimental data. The developed method can be used to predict the substrate reactivity and to interpret the specific nature of the enzyme catalysis.