Dominic Zichi

The use of aptamers in large arrays for molecular diagnostics.

BACKGROUND Aptamers are single-stranded oligonucleotides derived from an in vitro evolution protocol called systematic evolution of ligands by exponential enrichment (SELEX). They bind tightly and specifically to target molecules; most aptamers to proteins bind with Kds (equilibrium dissociation constant) in the range of 1 pM to 1 nM. METHODS AND RESULTS The SELEX protocol has been automated; therefore, hundreds to thousands of aptamers can be made in an economically feasible fashion. Blood and urine can be analyzed on chips that capture and quantitate proteins. SELEX has been adapted to the use of 5-bromo (5-Br) and 5-iodo (5-I) deoxyuridine residues. These halogenated bases can be specifically cross-linked to proteins. Selection pressure during in vitro evolution can be applied for both binding specificity and specific photo-cross-linkability. These are sufficiently independent parameters to allow one reagent, a photo-cross-linkable aptamer, to substitute for two reagents, the capture antibody and the detection antibody, in a typical sandwich array. After a cycle of binding, washing, cross-linking, and detergent washing, proteins will be specifically and covalently linked to their cognate aptamers. CONCLUSIONS Because no other proteins are present on the chips, protein-specific stain will now show a meaningful array of pixels on the chip. Learning algorithms and retrospective studies should lead to a robust, simple, diagnostic chip.

Let's get specific: the relationship between specificity and affinity

The factors that lead to high-affinity binding are a good fit between the surfaces of the two molecules in their ground state and charge complementarity. Exactly the same factors give high specificity for a target. We argue that selection for high-affinity binding automatically leads to highly specific binding. This principle can be used to simplify screening approaches aimed at generating useful drugs.

Post-SELEX combinatorial optimization of aptamers.

In vitro selection techniques provide a means of isolating nucleic acid ligands for binding to particular protein targets. Although most aptamers have quite high affinities for their target proteins, it has been shown that post-SELEX modification can result in further enhancement of binding affinity, as well as other desired properties. This has led to the current development of a more systematic approach to aptamer optimization using a combinatorial screening methodology.

A dynamical theory of unimolecular ionic dissociation reactions in polar solvents

A dynamical theory for the rates of unimolecular dissociations in polar solvents is constructed. Two classes of dissociation reactions, with dipolar and ionic transition states, are considered, and the theory is illustrated for a generalized continuum model water solvent. The rate of charge variation along the reaction coordinate is found to play a central role. Deviations from equilibrium solvation transition state theory predictions are found and discussed. Two nonequilibrium solvation regimes—nonadiabatic solvation and polarization caging—occur, and their appearance is connected to whether the solvated transition state has a reactant‐like or product‐like charge distribution.A dynamical theory for the rates of unimolecular dissociations in polar solvents is constructed. Two classes of dissociation reactions, with dipolar and ionic transition states, are considered, and the theory is illustrated for a generalized continuum model water solvent. The rate of charge variation along the reaction coordinate is found to play a central role. Deviations from equilibrium solvation transition state theory predictions are found and discussed. Two nonequilibrium solvation regimes—nonadiabatic solvation and polarization caging—occur, and their appearance is connected to whether the solvated transition state has a reactant‐like or product‐like charge distribution.

IDENTIFYING CONSENSUS PATTERNS AND SECONDARY STRUCTURE IN SELEX SEQUENCE SETS

Publisher Summary The general methodology presented for the analysis of SELEX (Systematic Evolution of Ligands by EXponential enrichment) isolated molecules relies on the power of consensus to guide multiple sequence alignment and identify common secondary structure. The information derived from consensus features among functionally related sequences is vastly greater than that obtained from single sequences. Computational tools have been developed that take advantage of consensus-derived information for the purpose of identifying the structural motifs responsible for the observed activity. Relatively high stringency is imposed on deriving the initial consensus patterns and structures, from which common motifs can be defined. Once a primary and secondary structural motif is obtained, sequences more distantly related may be identified by taking both sequence and structure into account for alignment. Ultimately, these data will be used to produce consensus 3D structural models for a set of molecules, with common functional properties. Such structural information is important for understanding and modifying the interactions critical for activity.

Theoretical Principles of In Vitro Selection Using Combinatorial Nucleic Acid Libraries

A new paradigm for drug discovery and biological research has developed from technologies that integrate combinatorial chemistry with rounds of selection and amplification, a technique called in vitro selection or systematic evolution of ligands by exponential enrichment (SELEX). This overview unit discusses nucleic acid libraries that can be used, affinity probability distributions, an equilibrium model for SELEX, and optimal conditions including concentrations and signal-to-noise ratios.

Environmental Dynamics and Electron Transfer Reactions

Recent theoretical and computer simulation work on the dynamics associated with electron transfer processes in polar solvents is described. This includes solvent relaxation subsequent to photo-induced charge transfer, adiabatic electron transfer rates, and the solvent influence on the electronic states relevant to electron transfers.

Non-equilibrium solvation in SN1 and SN2 reactions in polar solvents

In the standard transition-state theory (TST) view of the role of the polar solvent in ionic reactions, the solvent is implicitly assumed always to be in equilibrium with the intrinsic reaction system at each point along the reaction coordinate. However, if there is insufficient time for the solvent molecules to so equilibrate, there will be non-equilibrium solvation effects. These cause a breakdown in the TST predictions for the reaction rate. These effects are analytically described via van der Zwan–Hynes theory and Grote–Hynes theory and examined via a molecular-dynamics simulation of a model Cl–+CH3Cl SN2 reaction in water, and analytically for a model SN1 dissociation in water. Finally, a solution-phase reaction-path Hamiltonian theory is described to investigate anharmonic effects on model SN2 reactions.

Computer simulation study of electronic spectroscopy in water

Linear and nonlinear spectroscopy of liquid water is studied with molecular dynamics computer simulation techniques. The electronic structure variation of each solvent molecule with its local environment is effected via a truncated adiabatic basis-set description. By the inclusion of both linear and nonlinear electronic response, this accounts for the instantaneous adjustment of the water dipole moment and polarizability to the fluctuating local electric field. It also allows for the electronic relaxation effects associated with excitations through a mixing of different excited electronic configurations. By employing the TAB/10D potential model developed recently in our group, the electronic absorption, far-IR, depolarized Raman scattering and optical Kerr effect spectroscopy of water are examined under ambient conditions.