Combinatorial Chemistry on Solid Supports
In addition, enzymes can only produce polymers chemically and topologically similar to their natural products, which are not well-suited for all applications. An alternative strategy for expanding the chemical diversity of gene products exploits DNA-templated synthesis, where hybridization-induced proximity promotes covalent bond formation Gartner and Liu Each building block must be attached to an oligonucleotide, which is both expensive and labor intensive.
All chemistry must proceed under conditions compatible with DNA hybridization, ruling out many organic solvents, high pH, and high temperature. Finally, there may be a limitation to the number of steps that can be encoded by the proximity approach. While an impressive array of chemical reactions has been accomplished by this method Gartner et al.
DNA display is a general method for the in vitro selection of synthetic combinatorial chemistry libraries. The system is modular, so that chemistry and selection protocols can be easily changed. It can take advantage of existing combinatorial chemistry technology as well as chemical transformations previously carried out in the presence of unprotected DNA Gartner et al.
Solid-phase, solution-phase, enzymatic, and proximity effect reaction formats are all suitable. We have developed an extensive set of tools to adapt new chemistries for in vitro selection Halpin et al. In addition to diverse chemistries, many different library architectures are also possible. The library reported here was synthesized in six encoded steps with ten distinct building blocks per step. However, essentially any combinatorial scheme can be accommodated. As a first approximation, the highest possible fold enrichment per round of selection can be determined by considering its relationship to translation fidelity and the signal-to-noise ratio of the selection.
Fold enrichment E is defined as the geometric increase in the fraction of target molecules in a library that results from a single round of synthesis and selection. Fidelity F is defined as the fraction of genes recovered from a completed library synthesis that have been correctly translated. In most cases, the fold enrichment reduces to the simple expression at the right of Equation 1. Biological systems have such high fidelity that F can be considered to equal one.
However, the fidelity of chemical translation processes is the product of hybridization specificity and chemistry efficiency raised to the power of the number of steps. It is important to consider these parameters when adapting new chemistries and selections to the DNA display format. Equation 1 can help determine the minimum number of rounds required for library convergence, and thus the feasibility of a proposed in vitro selection experiment.
For example, a library synthesized with a fidelity of 0. If the library included 10 12 unique members, at least 12 rounds would be required to achieve convergence.
In addition to influencing convergence rates, fidelity also limits achievable library complexity. The maximum effective library complexity corresponds to the product of Avagadro's number, the moles of library, and the fidelity. Diversification between rounds of selection by recombination makes possible in vitro evolution of libraries with complexities exceeding the physical library size. Starting with a working population of compounds that sparsely sample a chemical space, molecules containing parts of an optimal molecular solution often have a selective advantage relative to siblings, and become enriched.
Subsequent recombination processes splice together fragments from the numerous partially optimal molecules to form a globally optimal molecule. Thus, the best structure is found, even if the odds were negligible that it existed in the initial working population. The same principle accounts for the striking success of gene shuffling in protein engineering Kurtzman et al.
COMBINATORIAL CHEMISTRY AND SOLID PHASE SYNTHESIS | sporleogesbadctrem.gq
Recombination of a DNA display library by DNA shuffling Stemmer , which was used here to diversify the initial DNA library Halpin and Harbury , would enable the in vitro evolution of synthetic libraries with complexities exceeding 10 DNA display enables the use of genetic tools such as complementation analysis and backcrossing to analyze small-molecule populations.
The approach can be used to study molecular evolution without potential biases resulting from experiments restricted to RNA, DNA, and peptide polymers.
A general scientific problem that will be directly addressed is the relationship between small-molecule library complexity and the quality of molecules discovered. With biopolymers, more complex libraries yield higher-affinity ligands Takahashi et al. This judgment is based on the paucity of viable drug candidates that have emerged from even the most complex combinatorial chemistry libraries.
Drug discovery would represent one important application for a small-molecule in vitro selection technology. A fast, inexpensive, and generally accessible procedure for the in vitro selection of druggable small-molecule libraries would accelerate the early stages of drug development. The nonnatural peptide chemistry in this work was developed as a proof of principle, but may nevertheless have practical applications in medicine. For example, the nonribosomal peptide drugs vancomycin and cyclosporin are a widely used antibiotic and immunosuppressant, respectively Walsh DNA display offers an immediate approach for the in vitro selection of general polyamide libraries that include such compounds Halpin et al.
Future extensions of DNA display include the development of massively parallel array-based splitting strategies for the in vitro selection of low-molecular-weight small-molecule libraries for example a library built in three synthetic steps with 10, building blocks per step. Beyond drug discovery, DNA display can be applied to the engineering of chemical switches, the discovery of transition metal catalysts for aqueous and nonbiological environments, and the identification of enzyme-specific ligands for activity-based profiling. Because the system is inexpensive, is easily implemented by a single individual, and requires only common laboratory equipment, in vitro selection and eventual evolution of large synthetic chemical populations should become a broadly accessible tool.
Solid-phase peptide synthesis was carried out as previously described Halpin et al. DNA was loaded onto the columns in 10 mM acetic acid, 0. To accomplish amino acid additions, columns were washed with 3 ml of DMF and subsequently incubated with Excess reagent was washed away with 3 ml DMF, and the coupling procedure was repeated.
Anhydride couplings followed the same procedure except that a 3-ml water wash was added after DNA loading to remove remaining acetic acid. For synthesis of libraries, a 2-ml PBS wash was added at the end of each amino acid coupling step to remove remaining anionic reagents. The electromobility shift assay was performed as previously described Hwang et al. No plasmid DNA was added to the samples. Antibody 3-E7 0. Then, preclear beads were pelleted by centrifugation and removed. High performance liquid chromatography analysis of DNA—peptide conjugates, synthesis of anticodon columns, hybridization and transfer of DNA, library assembly, ssDNA generation, and library isolation were performed as previously described Halpin and Harbury ; Halpin et al.
We thank Shivani Nautiyal and S. Jarrett Wrenn for critical reading of the manuscript and helpful discussions throughout the course of this work; Elizabeth Zuo for oligonucleotide synthesis and mass spectrometry analysis; and Mai Nguyen for oligonucleotide synthesis. DRH performed the experiments. Abstract 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.
Introduction Creation of molecular function represents a fundamental challenge. Results Strategy In vitro selection requires iterated rounds of three steps: conversion of genes to gene products, selection of gene products, and gene amplification Figure 1. Download: PPT.
Figure 2. Reduction to Practice We first developed a Sepharose-based resin derivatized with anticodon oligonucleotides complementary to codon sequences Halpin and Harbury In Vitro Selection of a Chemically Synthesized Library To test and validate our general strategy, we applied in vitro selection to a primarily nonnatural peptide library, with the goal of identifying a high-affinity ligand for the monoclonal antibody 3-E7 Meo et al.
Discussion Previous efforts to expand the scope of in vitro selection have utilized nonnatural bases or amino acids incorporated into DNA, RNA, and peptide libraries using polymerases and the ribosome Bittker et al.
In most cases, the fold enrichment reduces to the simple expression at the right of Equation 1 where f 0 denotes target gene fraction in the selection input and p denotes the probability of a nontarget gene being mistranslated to the target gene product. Prospectus DNA display enables the use of genetic tools such as complementation analysis and backcrossing to analyze small-molecule populations. Materials and Methods Materials.
Solid supports for combinatorial chemistry.
Electromobility shift assay. Acknowledgments We thank Shivani Nautiyal and S. References 1. J Am Chem Soc View Article Google Scholar 2. Anal Biochem — View Article Google Scholar 3. Curr Opin Chem Biol 6: — View Article Google Scholar 4. Breinbauer R, Vetter IR, Waldmann H From protein domains to drug candidates: Natural products as guiding principles in the design and synthesis of compound libraries. Angew Chem Int Ed Engl — View Article Google Scholar 5. View Article Google Scholar 6. Carpino LA, Han GY 9-fluorenylmethoxycarbonyl function, a new base-sensitive amino-protecting group.
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