Synthetic ligands discovered by in vitro selection

Abstract

The recognition and catalytic properties of biopolymers derive from an elegant evolutionary mechanism, whereby the genetic material encoding molecules with superior functional attributes survives a selective pressure and is propagated to subsequent generations. This process is routinely mimicked in vitro to generate nucleic-acid or peptide ligands and catalysts. Recent advances in DNA-programmed organic synthesis have raised the possibility that evolutionary strategies could also be used for small-molecule discovery, but the idea remains unproven. Here, using DNA-programmed combinatorial chemistry, a collection of 100 million distinct compounds is synthesized and subjected to selection for binding to the N-terminal SH3 domain of the proto-oncogene Crk. Over six generations, the molecular population converges to a small number of novel SH3 domain ligands. Remarkably, the hits bind with affinities similar to those of peptide SH3 ligands isolated from phage libraries of comparable complexity. The evolutionary approach has the potential to drastically simplify and accelerate small-molecule discovery.

Figure 1

DNA Display. A library of ssDNA molecules (top) is chemically translated into synthetic compound DNA conjugates. The DNA library is split into sub pools by hybridization of 20 base codons to complimentary oligonucleotide anticodons that are immobilized on separate columns (orange, cyan, pink bars). A distinct chemical transformation is carried out on each sub pool, resulting in the covalent attachment of a chemical building block to the DNA (orange, cyan, pink balls). The library is pooled and then split based on the next coding region (green, brown, yellow bars), and distinct chemical transformations are carried out for each sub pool. The process is iterated until the entire DNA sequence is read. Each codon can exist at only one coding region. The translated library is subjected to selection for a function of interest (binding to the immobilized grey widget), DNA linked to binders is amplified and used as input for the subsequent round of chemical translation. The entire process is repeated until the library converges. Enriched molecules are identified by DNA sequencing and assayed for function as pure compounds.

Figure 2

DNA encoded peptoid synthesis. (A) Peptoid structure, where R represents side chains. (B) DNA compatible submonomer peptoid synthesis. Each peptoid residue is constructed by chloroacetylation of a secondary amine followed by nucleophilic displacement with a primary amine. (C) HPLC analysis of submonomer peptoid coupling on DNA. A control 10 mer and a 5′ aminated 20 mer oligonucleotide starting; material were loaded onto DEAE Sepharose microcolumns, and subjected to chloroacetylation (red trace), or to chloroacetylation followed by nucleophilic displacement with propylamine (blue trace). The products were eluted and analyzed by reverse phase HPLC. Each peak was collected and its identity was verified by MALDI-MS. (D) HPLC analysis of polypeptoid synthesis on DNA. A 5′ animated 20 mer oligonucleotide (black trace) served as the starting material for an eight residue peptoid synthesis. The crude peptoid DNA product was analyzed by HPLC (red trace). The major peak (~30% yield) was confirmed as the intended product by MALDI MS (Observed: 7558, Expected: 7556).

Figure 3

Selection for N CrkSH3 binding. (A) The selection strategy utilizes an N-CrkSH3 protein (grey) with a prepended FLAG tag (white), as well as an anti FLAG antibody (black Y), and Pansorbin cells (yellow). After incubation of these components with a translated library, the Pansorbin cells are pelleted and washed. Ligand DNA conjugates are eluted by addition of an excess of an N-CrkSH3 binding peptide (black balls). (B) To measure the selection signal lo noise ratio, two peptide DNA conjugates were synthesized. One construct consisted of the known N-CrKSH3 peptide ligand YPPPALPPKRRR linked to a 340 mer dsDNA with a central BgII site (red). The other consisted of the peptide YGGFL linked to a 340 mer dsDNA with a central BamHI site (blue). When the ligand construct was diluted 1 to 1,000 into the non-ligand construct and amplified, BgII cutting was undetectable (lane 1). When this mixture was subjected to a mock selection that lacked N-CrkSH3 and was amplified, BgII cutting was undetectable (lane 2). When subjected to a selection with N-CrkSH3 included and amplified, the BgII containing DNA was enriched nearly 1,000 fold (lane 3).

Figure 4

In vitro selection of synthetic ligands that bind to N-CrkSH.3. (A) The library construct consisted of a 340 mer ssDNA molecule with eight coding regions (rainbow bars) and ten potential sequences at each region (10⁸ possible library members). The monomers used at each coding region are indicated. Each monomer is assigned an arbitrary letter code for convenience. The coding regions are numbered according to the N to C sequence of the peptoid product, but are read C to N during chemical translation. (B) The percent of input DNA recovered in the selection eluate is plotted with respect to selection round. DNA concentrations were determined by quantitative PCR. Error bars represent standard deviations of repeat measurements. (C) Clustergram of the sixth generation peptoid sequences. Red indicates sequence similarity. A representative peptoid from each major cluster (numbered in white relief) was synthesized for binding assays. ’Vector’ Indicates sequences derived from the cloning vector, and not from the library.

Figure 5

(A) Intrinsic tryplophan fluorescence based binding assay. The fraction of N-CrkSH3 that is bound by ligand is plotted as a function of Crktoid 8 concentration (red points). The data points are averages of three separate experiments, with standard deviations shown, The Kd value was determined by filling the averaged data to a binding curve (black line). (B) Sequences and Kd values of resynthesized peptoids, named according to cluster and sorted by affinity. The numbers above the sequences refer to the coding positions as in Figure 4. Side chains have been categorized as small (yellow), bulky (green), singly positively charged {light blue), and multiply positively charged (dark blue). (C) Chemical structures of the validated ligands sorted by decreasing affinity.