Interaction-dependent PCR: Multiplexed identification of ligand-target pairs from DNA-encoded ligand/target libraries in a single solution-phase experiment
The rapid identification of ligands to an ever-expanding number of biological targets of interest is a major technological challenge in the life sciences. Two fundamental limitations to existing target-oriented high-throughput screening methods are (i) the requirement that each target must successively be assayed against libraries of potential ligands (limits assay throughput), and (ii) the general reliance on immobilized targets or ligands adds immobilization, washing, and/or elution steps and is a source of artifacts. A solution-phase method to simultaneously reveal all ligand-target binding pairs from a single solution containing libraries of ligands and libraries of targets could overcome both limitations and significantly increase the efficiency and effectiveness of target oriented screening efforts. Researchers in the laboratory of Professor David Liu have developed such a method, interaction-dependent PCR (IDPCR).
Innovations and Advantages
IDPCR is based on the melting temperature difference between duplex DNA formed intramolecularly versus intermolecularly. Binding of a target to its ligand would increase the effective molarity of single-stranded DNA oligonucleotides linked to the target and ligand, promoting duplex formation between complementary regions on each strand that are otherwise too short to hybridize. The resulting hairpin thus serves as a starting point for primer extension. Crucially, only the newly extended hairpin contains in a single DNA strand two primer (or primer-binding) sequences that enable subsequent PCR amplification. IDPCR therefore results in the selective amplification of those DNA sequences previously attached to, and therefore encoding, ligand-target pairs.
In a model multiplex selection of a 261-member DNA-encoded ligand library with a 259-member DNA-encoded target library, designed to contain only five true ligand-target pairs out of 67,599 possible interactions, all five known ligand-target pairs were significantly enriched relative to control, with only 3 false positive signals (see figure).
Overview of IDPCR: (a) A model library of DNA-encoded ligands mixed with a model library of DNA-encoded targets allows multiplexed detection of binding pairs. (b) IDPCR was used to perform a model selection on an equimolar 261-member DNA-ligand library and an equimolar 259-member DNA-target library containing five known protein-ligand pairs out of 67,599 possible combinations. For each protein target, the most highly enriched sequences (blue bars) relative to a control lacking proteins corresponded to the known protein-ligand pairs, labeled A-E in the plot. A: biotin + streptavidin (SA); B: desthiobiotin + SA; C: Gly-Leu-4-carboxy benzene sulfonamide + carbonic anhydrase II (CA), D: carboxy benzene sulfonamide + CA; E: trypsin +antipain.
IDPCR represents a general method for selectively amplifying DNA sequences encoding ligand-target pairs. IDPCR can be applied to a wide variety of targets and potential ligands and can identify ligand-target pairs from libraries of small molecules and libraries of targets in a single solution. IDPCR is highly sensitive, takes place entirely in the solution phase, and can be performed in a few hours using routine equipment. A variation of this method, reactivity-dependent PCR, has been used for the multiplexed identification of molecules that undergo bond formation or cleavage. IDPCR will significantly enhance efforts to discover new ligands and targets, to reveal target-binding specificities of small molecules, and to detect low-abundance analytes.
Intellectual Property Status: A patent application is pending.
Interaction-Dependent PCR: Identification of Ligand-Target Pairs from Libraries of Ligands and Libraries of Targets in a Single Solution-Phase Experiment, McGregor LM, Gorin DJ, Dumelin CE, Liu DR J. Am. Chem. Soc. 132, 15522-15524 (2010).
Reactivity-Dependent PCR: Direct, Solution-Phase In Vitro Selection for Bond Formation, Gorin DJ, Kamlet AS, Liu DR J. Am. Chem. Soc. 131, 9189-9191 (2009).
Dumelin, Christoph E.
Gorin, David J.
Kamlet, Adam S.
Liu, David R.
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Reference Harvard Case #3580