DNA display II. Genetic manipulation of combinatorial chemistry libraries for small-molecule evolution

Biological in vitro selection techniques, such as RNA aptamer methods and mRNA display, have proven to be powerful approaches for engineering molecules with novel functions. These techniques are based on iterative amplification of biopolymer libraries, interposed by selection for a desired functional property. Rare, promising compounds are enriched over multiple generations of a constantly replicating molecular population, and subsequently identified. The restriction of such methods to DNA, RNA, and polypeptides precludes their use for small-molecule discovery. To overcome this limitation, we have directed the synthesis of combinatorial chemistry libraries with DNA "genes," making possible iterative amplification of a nonbiological molecular species. By differential hybridization during the course of a traditional split-and-pool combinatorial synthesis, the DNA sequence of each gene is read out and translated into a unique small-molecule structure. This "chemical translation" provides practical access to synthetic compound populations 1 million-fold more complex than state-of-the-art combinatorial libraries. We carried out an in vitro selection experiment (iterated chemical translation, selection, and amplification) on a library of 10(6) nonnatural peptides. The library converged over three generations to a high-affinity protein ligand. The ability to genetically encode diverse classes of synthetic transformations enables the in vitro selection and potential evolution of an essentially limitless collection of compound families, opening new avenues to drug discovery, catalyst design, and the development of a materials science "biology."     

Automated design of specificity in molecular recognition

Specific protein-protein interactions are crucial in signaling networks and for the assembly of multi-protein complexes, and represent a challenging goal for protein design. Optimizing interaction specificity requires both positive design, the stabilization of a desired interaction, and negative design, the destabilization of undesired interactions. Currently, no automated protein-design algorithms use explicit negative design to guide a sequence search. We describe a multi-state framework for engineering specificity that selects sequences maximizing the transfer free energy of a protein from a target conformation to a set of undesired competitor conformations. To test the multi-state framework, we engineered coiled-coil interfaces that direct the formation of either homodimers or heterodimers. The algorithm identified three specificity motifs that have not been observed in naturally occurring coiled coils. In all cases, experimental results confirm the predicted specificities.     

Potential energy functions for protein design

Different potential energy functions have predominated in protein dynamics simulations, protein design calculations, and protein structure prediction. Clearly, the same physics applies in all three cases. The differences in potential energy functions reflect differences in how the calculations are performed. With improvements in computer power and algorithms, the same potential energy function should be applicable to all three problems. In this review, we examine energy functions currently used for protein design, and look to the molecular mechanics field for advances that could be used in the next generation of design algorithms. In particular, we focus on improved models of the hydrophobic effect, polarization and hydrogen bonding.     

Mesofluidic devices for DNA-programmed combinatorial chemistry

Hybrid combinatorial chemistry strategies that use DNA as an information-carrying medium are proving to be powerful tools for molecular discovery. In order to extend these efforts, we present a highly parallel format for DNA-programmed chemical library synthesis. The new format uses a standard microwell plate footprint and is compatible with commercially available automation technology. It can accommodate a wide variety of combinatorial synthetic schemes with up to 384 different building blocks per chemical step. We demonstrate that fluidic routing of DNA populations in the highly parallel format occurs with excellent specificity, and that chemistry on DNA arrayed into 384 well plates proceeds robustly, two requirements for the high-fidelity translation and efficient in vitro evolution of small molecules.     

Die Flora der Drauterrassen in Unterkärnten

Ohne Zusammenfassung     

The N-Terminal Amino Acids of Human Plasma Proteins

A study has been made of the N-terminal amino acid pattern of human plasma proteins under normal and pathological conditions. The normal pattern shows the following N-terminal amino acids in order of diminishing quantities: aspartic acid, glutamic acid, valine, alanine, tyrosine, leucines, and glycine. In healthy individuals this pattern is qualitatively stable with moderate quantitative differences between individuals. On the other hand radical quantitative changes in the pattern have been observed under pathological conditions.