Use of self-assembled monolayers to probe the structure of a target molecule

Abstract

Weak binding motifs were transformed into a high affinity ligand surface by using a heterologous self-assembled monolayer (SAM) as a rigid scaffold to present discrete binding moieties, in a controlled geometry, to a target molecule. At a critical ligand density, the discrete binding moieties simulated a multivalent ligand and promoted high-affinity, cooperative binding of the target molecule. Statistical calculations were applied to SAM components in solution and gold-sulfur packing dimensions to extract the inter-ligand-distance within the SAM. This distance information is valuable to the rational design of multivalent drugs.

Description

RELATED APPLICATIONS [0001] This application is a continuation of U.S. patent application Ser. No. 09 / 324,258, filed Jun. 2, 1999, which claims priority to U.S. Provisional Patent Application No. 60 / 087,766, filed on Jun. 2, 1998.STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT [0002] This work was supported, in part, by NIH Grants 5T32EM-07598-18 and GM-32308. The government of the United States of America may have some rights in this invention.FIELD OF INVENTION [0003] The present invention relates to the use of self-assembled monolayers attached to surfaces for the detection and probing of target molecule structure and function. BACKGROUND OF THE INVENTION [0004] Combinatorial chemistry techniques are used to synthesize diverse “libraries” of unique chemical compounds. These small molecule libraries often yield drug candidates that are capable of binding a specific biological target but because of their small size and relative simple ch...

AI Technical Summary

Benefits of technology

[0008] Self-assembled monolayers are used as a rigid 2-dimensional matrix for presenting binding moieties, at varying distances from each other, to a target molecule. Two-component SAMs incorporate an inert spacer molecule and a biospecific molecule that can directly or indirectly present a binding moiety to a target molecule. The distance between the biospecific molecules in the array, the ligand density, is controlled by manipulating the concentrations of the two component thiols in solution before deposition onto gold. The affinity of the interaction between the surface immobilized ligands and the multivalent target molecule is monitored as a function of ligand density. The lowest ligand surface density that elicits a jump in affinity for the target molecule contains the critical information needed to extract the distance between binding sites on the target molecule. The dimensions of the hexagonal tiling pattern formed when the sulfurs from the thiols bind to gold solid are known. Therefore, Poisson statistics can be used to infer the distance between surface immobilized ligands, and thus the inter-binding-site distance on the target molecule, from the concentrations of the thiols in solution. Further, the gold surface itself and the attached SAM can be used as a scaffold to present binding moieties, in a controlled, higher affinity geometry, to a target molecule.

[0009] In a preferred embodiment, SAMs are generated that incorporate two thiol types: 1) an inert tri-ethylene glycol-terminated thiol; and 2) a nitrilo tri-acetic acid (NTA) terminated thiol that when complexed with Ni, captures histidine-tagged proteins or peptides. The density of NTA-thiol within the SAM is varied to present varying densities of a histidine-tagged binding moiety to a multi-valent target molecule. The affinity of the interaction is plotted as a function of ligand density within the SAM. A dramatic increase in the binding affinity occurs at a critical surface density when the presented ligands are close enough to each other to simultaneously bind to a common target molecule. The solution concentrations of the two thiol types and the dimensions of the tiling pattern that the thiols form on the gold substrate are input into Poisson distribution equations to extract the probable distance between binding sites on a target molecule.

Problems solved by technology

These low affinity interactions cannot adequately compete with larger more diverse natural ligands, like proteins and protein complexes, and thus provide little therapeutic value.

The problem with developing a natural product for the drug marked is that they are large and chemically complicated, which means that elaborate and expensive schemes for their synthesis must be developed.

Identifying a synthetic scheme that is commercially feasible is a technical challenge that at best takes years and millions of dollars to accomplish and at worst cannot be done.

The problem with this logic is that the enthalpic advantage of the additional binding energy is offset by the large entropic energy cost of ordering the connected binding moieties.

This process is time-consuming (years) and expensive.

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