Chemical and Genetic Wrappers for Improved Phage and RNA Display

Abstract

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 non-specificity to positively charged target molecules, which represent an estimated 35% of proteins in the human proteome. 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. First, a genetic strategy introduced a short charge neutralizing peptide into the solvent-exposed, negatively charged phage coat. The modified phage (KO7+) reduced or eliminated non-specific binding to the problematic high pI proteins. In the second, chemical approach, oligolysine wrappers for phage and total RNA blocked non-specific interactions. For phage display applications, the peptides Lysn (where n = 16 to 24) emerged as optimal for wrapping the phage. Lys8, however, provided effective wrappers for RNA binding in assays against the RNA binding protein HIV-1 Vif. The oligolysine peptides blocked non-specific binding to allow successful selections, screens, and assays with five previously unworkable protein targets.

Phage display and molecular imaging: expanding fields of vision in living subjects

“…However, false positives may arise from retention of clones that bind non-specifically or from clones with genetic advantages for propagation that are not related to target binding (Brammer et al 2008). A recent report described an approach to address issues with non-specific charge interactions by genetically modifying phage coat proteins or using chemical agents to suppress non-specific interactions that occur between the negative charge on the phage coat surface with positively charged targets (Lamboy et al 2008). Phage clones recovered from library selections are then individually validated for target binding; yet, this is usually a time-consuming step that can strongly benefit from new methods to rapidly prioritize hits.…”

Chemical modulation of M13 bacteriophage and its functional opportunities for nanomedicine

“…25 The viral capsid is aligned along the shaft and is composed of 2,700 copies of pVIII and ~5 copies of minor coat proteins pIII, pVI, pIX, and pVII located at either end. 21,22 The 50-residue pVIII (98% by mass) is composed of three distinct domains, namely, a negatively charged hydrophilic N-terminal domain (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), an intermediate hydrophobic domain (21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39), and a positively charged domain (40)(41)(42)(43)(44)(45)(46)…”

“…Lamboy et al 45 attempted to reduce nonspecific binding of positively charged proteins to the native negatively charged surface of phage for improved phage display system development. In order to neutralize the phage surface, the authors had either genetically engineered the phage to display charge-neutralizing peptides on the solvent-exposed surface or blocked the surface with oligolysine (eg, Lys8) against highly basic proteins in the proteome.…”

Engineering chemically modified viruses for prostate cancer cell recognition

“…The high negative charge on the phage surface allows non-covalent wrapping with cationic peptides and polymers. 29,30 Linking these wrappers to recognition ligands opens new routes to greater sensitivity and specificity for target analytes. The peptide ligands can be chemically synthesized and fused to an oligolysine peptide (K 14 ), which ‘wraps’ around the virus particle through complementary electrostatic interactions.…”