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. 2014;5(1):e27896.
doi: 10.4161/adna.27896.

Universal strategies for the DNA-encoding of libraries of small molecules using the chemical ligation of oligonucleotide tags

Alexander Litovchick  1 , Matthew A Clark, Anthony D Keefe
Affiliations

Universal strategies for the DNA-encoding of libraries of small molecules using the chemical ligation of oligonucleotide tags

Alexander Litovchick et al. Artif DNA PNA XNA. 2014.

Abstract

The affinity-mediated selection of large libraries of DNA-encoded small molecules is increasingly being used to initiate drug discovery programs. We present universal methods for the encoding of such libraries using the chemical ligation of oligonucleotides. These methods may be used to record the chemical history of individual library members during combinatorial synthesis processes. We demonstrate three different chemical ligation methods as examples of information recording processes (writing) for such libraries and two different cDNA-generation methods as examples of information retrieval processes (reading) from such libraries. The example writing methods include uncatalyzed and Cu(I)-catalyzed alkyne-azide cycloadditions and a novel photochemical thymidine-psoralen cycloaddition. The first reading method "relay primer-dependent bypass" utilizes a relay primer that hybridizes across a chemical ligation junction embedded in a fixed-sequence and is extended at its 3'-terminus prior to ligation to adjacent oligonucleotides. The second reading method "repeat-dependent bypass" utilizes chemical ligation junctions that are flanked by repeated sequences. The upstream repeat is copied prior to a rearrangement event during which the 3'-terminus of the cDNA hybridizes to the downstream repeat and polymerization continues. In principle these reading methods may be used with any ligation chemistry and offer universal strategies for the encoding (writing) and interpretation (reading) of DNA-encoded chemical libraries.

Keywords: chemical ligation; click chemistry; combinatorial chemistry; drug discovery; in vitro selection; modified nucleotide; photochemistry; polymerase; template-dependent polymerization; unimolecular information recording and retrieval.

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Figures

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Figure 1. Mechanism of cDNA generation from chemically ligated tags using relay primer-mediated bypass on a photochemically ligated non-traversable template. A terminal primer is annealed to the 3′-terminus of the chemically ligated oligonucleotide and a second 5′-monophosphorylated primer (relay primer) is annealed across the chemical ligation junction. Extension of both primers and ligation generates full-length cDNA including potentially encoding sequences from both upstream and downstream of the photochemical ligation junction.
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Figure 2. Psoralen-mediated photochemical oligonucleotide ligation. (A) The photochemical ligation of a 3′-thymidine oligonucleotide to a 5′-psoralen oligonucleotide after co-annealing to a complementary splint oligonucleotide (not shown). The stereochemistry of the thymidine-psoralen adduct may be either cis or trans. (B) Denaturing PAGE analysis of the time-course of the photochemical reaction that results in the ligation of a 3′-thymidine oligonucleotide to a 5′-psoralen oligonucleotide with illumination at 365 nm, the gel is imaged by UV shadowing of a fluorescent plate. The appearance of the photochemical ligation product is indicated with a conversion of 50% occurring between two and four hours of illumination.
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Figure 3. cDNA generation from chemically ligated tags using relay primer-mediated bypass on a photochemically ligated non-traversable template. (A) LC analysis showing approximately equal amounts of two primer-extension products after purification with detection at 495 nm. (B) Deconvoluted MS analysis of mixed primer-extension products showing two principal components with masses of 15,172 Da and 11,157 Da which correspond to full-length cDNA minus one dT, and primer extension up to the photochemical ligation junction respectively.
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Figure 4. Proposed mechanism for the generation of cDNA from chemically ligated tags using repeat-dependent bypass. A terminal primer is annealed to the 3′-terminus of a chemically ligated oligonucleotide with a repeated sequence either side of the chemical ligation junction. Upon extension of this primer with a polymerase the newly generated cDNA stalls at the chemical ligation junction having copied the first of the repeated sequences. Rearrangement occurs with loop formation and the slippage of the terminus of the cDNA to become hybridized to the second instance of the repeated sequence, whereupon extension resumes and generates a full-length cDNA except for the deletion of one of the repeated sequences.
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Figure 5A and B. (A) Controlled concatenation of three oligonucleotides using successive Cu(I)-free and Cu(I)-catalyzed chemical ligation by triazole formation. First the central oligonucleotide (7) is chemically ligated to (8) using the copper-free reaction of the azide group in (7) with the strained alkyne in (8), thereby preserving the terminal alkyne in (7) which is subsequently reacted in a Cu(I)-catalyzed reaction with the azide in the subsequently added third oligonucleotide (9). (B) LC time-course of Cu(I)-free conjugation of (7) with (8) using absorbance at 260 nm.
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Figure 5C–E. (C) LC time-course of Cu(I)-catalyzed conjugation of (9) with the conjugate of (7) and (8) using absorbance at 260 nm. (D) LC analysis of gel-purified doubly-conjugated products (7)+(8)+(9) and (7)+(10)+(11) using absorbance at 260 nm. (E) Deconvoluted MS of gel-purified doubly-conjugated products (7)+(8)+(9) “short-short” and (7)+(10)+(11) “long-long.”
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Figure 6. Demonstration of the fidelity of cDNA generation from chemically ligated tags using repeat-dependent bypass. A pair of doubly-conjugated oligonucleotides was prepared - one by the chemical ligation of two short oligonucleotides to a central oligonucleotide and the other by the chemical ligation of two long oligonucleotides to the same central oligonucleotide. The corresponding junction in each conjugate was flanked by the same repeated sequence. cDNA generation and subsequent PCR and electrophoresis using ethidium-stained agarose indicates that each of the conjugates may be amplified using relay primer-mediated bypass and that amplification occurs without the occurrence of homology-mediated shuffling, as indicated by the presence of bands with mobilities corresponding to amplification products containing “long-long” and “short-short” tag combinations, and the absence of the chimeric amplification products “long-short” and “short-long.”
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Scheme 1. Sequences and chemical modifications of oligodeoxynucleotides used in this study.
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