Aggregation of DNA oligomers with a neutral polymer facilitates DNA solubilization in organic solvents for DNA-encoded chemistry

Abstract

Chemical diversification of DNA-conjugated substrates is key in DNA-encoded library (DEL) synthesis and other nucleic acid-based technologies. One major challenge to the translation of synthesis methods to DNA-tagged substrates is the lack of solubility of the highly charged DNA oligomer in most organic solvents. A neutral acrylate block copolymer, devoid of any canonical nucleic acid-binding structure, tightly interacted with DNA oligonucleotides in their ammonium form, and solubilized them in nonpolar solvents such as dichloromethane, chloroform and toluene. The ternary DNA-copolymer-ammonium salt interactions led to the formation of aggregates in organic solvents whose size correlated with DNA oligomer length. This method for DNA solubilization was successfully applied to diversify DNA-tagged starting materials by three isocyanide multicomponent reactions (IMCR) with broad scope and excellent yields. The copolymer does not require tailored DNA conjugates and solubilized DNA oligomers of up to 80 nucleotides length. It will likely broaden the toolbox of DEL-compatible synthesis methods well beyond IMCR chemistry and it has application potential in other nucleic acid-based technologies that require a broadened solvent scope for nucleic acid conjugate synthesis.

Fig. 1. Strategies for DNA-encoded chemistry in organic solvents. (A) DNA-encoded split-pool synthesis and published clinical candidates that originated from DEL screens. (B) Immobilization of DNA tags on ion exchange resins. (C) Solubilization of DNA tags in polar organic solvents as tight ion pairs with cationic surfactants. (D) Encoded chemistry on a PEG polymer–DNA conjugate. (E) This work: solubilization of DNA tags as ammonium salts in organic solvents with an amphiphilic block copolymer and application to three isocyanide multicomponent reactions (IMCR). (F) Approved drugs synthesized by IMCRs.

Fig. 2. DNA solubilization experiments and structural characterization of DNA–copolymer aggregates in organic solvents. (A) DNA oligomers were solubilized by the copolymer in organic solvents. (B) Photographical pictures of FAM-labelled 20mer DNA oligomer solubilized by the copolymer in various organic solvents: (I) DNA + copolymer + Et₃NH⁺ in toluene; (II) DNA in DCM w/o copolymer; (III) DNA + copolymer + Et₃NH⁺ as counterion in DCM; (IV) DNA in CHCl₃ w/o copolymer; (V) DNA + copolymer + Et₃NH⁺ as counterion in CHCl₃; (VI) DNA + copolymer + NH₄⁺ as counterion in DCM. (C) ¹H NMR spectra of a hexathymidine (hexT) DNA oligomer in D₂O (lower panel), a mixture of hexT and the copolymer (middle panel), and the copolymer alone (upper panel) in CDCl₃. The spectra were scaled for better comparability. Signal assignment is indicated and well separated signals for the DOSY analysis, are highlighted by colored boxes. (D) Structural characterization of DNA–copolymer mixtures by transmission electron microscopy (TEM). (I) TEM image of 20mer DNA (5 nmol in 300 μL) and 30 equiv. copolymer in water; (II) TEM image of 20mer DNA (5 nmol in 300 μL) and 30 equiv. copolymer in chloroform; (III) TEM image of 80mer DNA (5 nmol in 300 μL) and 30 equiv. polymer in chloroform.

Fig. 3. Scope of the Ugi four-component reaction on 10mer TC-DNA. Reaction conditions: TC-1 (3 nmol) and copolymer (90 nmol) and primary amine 2 (6 μmol) in MeOH/CHCl₃ (50 μL; 3:1, v/v) for 3 h at rt. Addition of isocyanide 3 (6 μmol) and carboxylic acid 4 (6 μmol). The reaction was run for 16 h at 50 °C.

Fig. 4. Scope of the Ugi-azide four-component reaction on 10mer TC-DNA. Reaction conditions: TC-1 (3 nmol) and copolymer (90 nmol) and secondary amine 6 (6 μmol) in MeOH/CHCl₃ (50 μL; 3:1, v/v) for 3 h at rt. Addition of isocyanide 3 (6 μmol) and TMSN₃7 (6 μmol). The reaction was run for 16 h at 50 °C.

Fig. 5. Scope of the Groebke–Blackburn–Bienaymé three-component reaction on 10mer TC-DNA. Reaction conditions: TC-1 (3 nmol) and copolymer (90 nmol) and heteroaromatic amine 9 (6 μmol) in MeOH/CHCl₃ (80 μL; 7:1, v/v) for 6 h at rt. Addition of isocyanide 3 (6 μmol) and acetic acid (0.8 μL; c = 1 vol%). The reaction was run for 16 h at 25 °C.

Fig. 6. DNA-scope of multicomponent reactions, reactions with DNA-tagged mixtures and DNA compatibility assessment. DNA sequences: ATGC: 5′-(C6)aminolinker-CTACGTATGTGACC; 7dATC: 5′-CTACATAXCTATCC, X denotes a 5-triazolylthymine residue as linker to the barcoded compound. (A–C) Synthesis of DNA barcode-tagged product mixtures by Groebke–Blackburn–Bienaymé (A), Ugi-4-component (B), and Ugi-azide (C) reactions with mixtures of DNA-tagged aldehydes. As barcode we used a chemically stabilized 14mer DNA 7dATC. (D) DNA damage assessment after the three IMCRs on 7dATC-barcode by ligation, barcode amplification by PCR and Sanger sequencing of the amplicon. Analysis of ligated products by gel electrophoresis: (i) DNA ladder; (ii) 7dATC-5a; (iii) product of barcode ligation and barcode PCR of 7dATC-5a [U-4CR; 178 bp]; (iv) 7dATC-8a; (v) product of barcode ligation and barcode PCR of 7dATC-8a [UA-4CR, 178 bp]; (vi) 7dATC-10c; (vii) product of barcode ligation and barcode PCR of 7dATC-10c [GBB-3CR, 178 bp]; (viii) DNA ladder.