DNA-templated organic synthesis and selection of a library of macrocycles

Abstract

The translation of nucleic acid libraries into corresponding synthetic compounds would enable selection and amplification principles to be applied to man-made molecules. We used multistep DNA-templated organic synthesis to translate libraries of DNA sequences, each containing three "codons," into libraries of sequence-programmed synthetic small-molecule macrocycles. The resulting DNA-macrocycle conjugates were subjected to in vitro selections for protein affinity. The identity of a single macrocycle possessing known target protein affinity was inferred through the sequence of the amplified DNA template surviving the selection. This work represents the translation, selection, and amplification of libraries of nucleic acids encoding synthetic small molecules rather than biological macromolecules.

Fig. 1

Scheme for the translation, selection, and amplification of libraries of DNA templates encoding synthetic small molecules. When the number of different possible library structures approaches or exceeds the number generated, template diversification after selection can be added to evolve the pool of synthetic molecules toward structures possessing desired properties. X is a starting material common to all library members.

Fig. 2

(A) DNA-templated macrocycle library synthesis scheme. R is NHCH₃ where R is NHCH₃ or tryptamine; Ar, –(p-C₆H₄)–. The macrocyclization reaction is confirmed to give predominantly trans alkene stereochemistry for one library member (fig. S4) (13) but may give other outcomes for different macrocyclic structures. (B) Template and reagents used in the DNA-templated synthesis of 8a. (C) Denaturing PAGE of each step in the DNA-templated synthesis of 8a. Lane h is the product of a DNA-templated thiol addition to the product shown in lane g, confirming the formation of the fumaramide group during macrocyclization.

Fig. 3

(A) Building blocks and anticodons used in reagents for DNA-templated macrocycle library synthesis. The variable regions within each anticodon are underlined. The NlaIII cleavage site within template 1e is shown in lower case. (B) Representative DNA templates used in macrocycle library synthesis. Templates 1a to 1e (R is NHCH₃ or tryptamine) collectively call for each of the reagents in (A). (C) Denaturing PAGE analysis confirming the sequence specificity of each template-reagent combination used in the macrocycle library synthesis. The listed template and reagent(s) were combined under the conditions shown in Fig. 2A, and the reactions were analyzed before reagent-linker cleavage. Products appear as bands of higher molecular weight above templates.

Fig. 4

MALDI-TOF mass spectrometric analyses (M–H negative ion mode) of (A) the multistep DNA-templated synthesis of 8a starting from 1a (R is NHCH₃), (B) the translation of four templates (1a to 1d; R is tryptamine) into four corresponding macro-cycles (8a to 8d), and (C) step two of the translation of 65 templates (the 64 templates containing all possible combinations of codons complementing 2a to 2d, 3a to 3d, 4a to 4d, plus template 1e) into 65 corresponding macrocycles. (D) Analysis of the DNA-templated 65-member macrocyclic fumaramide library by denaturing PAGE (lanes a to c) and agarose gel electrophoresis (lanes d to g). Lane a, DNA-linked thiol reagent complementing the constant 5′ region of all macro-cycle template sequences; lane b, the 65-member library of template-linked macro-cycles (8); lane c, the library after DNA-templated thiol addition, confirming the presence of the fumaramide group formed during macrocyclization; lane d, NlaIII digestion of PCR-amplified templates from the 65-member macrocycle library before selection; lanes e and f, NlaIII digestion of PCR-amplified templates from the 65-member macrocycle library after one and two rounds of selection for carbonic anhydrase affinity, respectively; lane g, NlaIII digestion of authentic PCR-amplified template 1e that directs the synthesis of 8e.