Chemical and Genetic Wrappers for Improved Phage and RNA Display
| Autor: | Robert M. Corn, Pilgrim J. Jackson, Sara K. Avrantinis, Phillip Y. Tam, Lucie S. Lee, Jorge A. Lamboy, Hye Jin Lee, Gregory A. Weiss |
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| Rok vydání: | 2008 |
| Předmět: |
Phage display
Molecular Sequence Data Enzyme-Linked Immunosorbent Assay Computational biology Biology Ligands Biochemistry Article Peptide Library vif Gene Products Human Immunodeficiency Virus False positive paradox Bacteriophages Amino Acid Sequence RNA Messenger Molecular Biology Genetics Nonspecific binding Messenger RNA Deoxyribonucleases Lysine Organic Chemistry RNA-Binding Proteins RNA Mutagenesis Ribosome display Molecular Medicine Bacteriophage M13 Protein Binding |
| Zdroj: | ChemBioChem. 9:2846-2852 |
| ISSN: | 1439-7633 1439-4227 |
| DOI: | 10.1002/cbic.200800366 |
| Popis: | An Achilles heel inherent to all molecular display formats, background binding between target and display system introduces false positives into screens and selections. For example, the negatively charged surfaces of phage, mRNA, and ribosome display systems bind with unacceptably high nonspecificity to positively charged target molecules, which represent an estimated 35% of proteins in the human proteome. Here we report the first systematic attempt to understand why a broad class of molecular display selections fail, and then solve the underlying problem for both phage and RNA display. Firstly, a genetic strategy was used to introduce a short, charge-neutralizing peptide into the solvent-exposed, negatively charged phage coat. The modified phage (KO7(+)) reduced or eliminated nonspecific binding to the problematic high-pI proteins. In the second, chemical approach, nonspecific interactions were blocked by oligolysine wrappers in the cases of phage and total RNA. For phage display applications, the peptides Lys(n) (where n=16 to 24) emerged as optimal for wrapping the phage. Lys(8), however, provided effective wrappers for RNA binding in assays against the RNA binding protein HIV-1 Vif. The oligolysine peptides blocked nonspecific binding to allow successful selections, screens, and assays with five previously unworkable protein targets. |
| Databáze: | OpenAIRE |
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