TY - JOUR
T1 - An ELISA-based platform for rapid identification of structure-dependent nucleic acid–protein interactions detects novel DNA triplex interactors
AU - Economos, Nicholas G.
AU - Thapar, Upasna
AU - Balasubramanian, Nanda
AU - Karras, Georgios I.
AU - Glazer, Peter M.
N1 - Publisher Copyright:
© 2022 The Authors
PY - 2022/10
Y1 - 2022/10
N2 - Unusual nucleic acid structures play vital roles as intermediates in many cellular processes and, in the case of peptide nucleic acid (PNA)–mediated triplexes, are leveraged as tools for therapeutic gene editing. However, due to their transient nature, an understanding of the factors that interact with and process dynamic nucleic acid structures remains limited. Here, we developed snapELISA (structure-specific nucleic acid-binding protein ELISA), a rapid high-throughput platform to interrogate and compare up to 2688 parallel nucleic acid structure–protein interactions in vitro. We applied this system to both triplex-forming oligonucleotide–induced DNA triplexes and DNA-bound PNA heterotriplexes to describe the identification of previously known and novel interactors for both structures. For PNA heterotriplex recognition analyses, snapELISA identified factors implicated in nucleotide excision repair (XPA, XPC), single-strand annealing repair (RAD52), and recombination intermediate structure binding (TOP3A, BLM, MUS81). We went on to validate selected factor localization to genome-targeted PNA structures within clinically relevant loci in human cells. Surprisingly, these results demonstrated XRCC5 localization to PNA triplex-forming sites in the genome, suggesting the presence of a double-strand break intermediate. These results describe a powerful comparative approach for identifying structure-specific nucleic acid interactions and expand our understanding of the mechanisms of triplex structure recognition and repair.
AB - Unusual nucleic acid structures play vital roles as intermediates in many cellular processes and, in the case of peptide nucleic acid (PNA)–mediated triplexes, are leveraged as tools for therapeutic gene editing. However, due to their transient nature, an understanding of the factors that interact with and process dynamic nucleic acid structures remains limited. Here, we developed snapELISA (structure-specific nucleic acid-binding protein ELISA), a rapid high-throughput platform to interrogate and compare up to 2688 parallel nucleic acid structure–protein interactions in vitro. We applied this system to both triplex-forming oligonucleotide–induced DNA triplexes and DNA-bound PNA heterotriplexes to describe the identification of previously known and novel interactors for both structures. For PNA heterotriplex recognition analyses, snapELISA identified factors implicated in nucleotide excision repair (XPA, XPC), single-strand annealing repair (RAD52), and recombination intermediate structure binding (TOP3A, BLM, MUS81). We went on to validate selected factor localization to genome-targeted PNA structures within clinically relevant loci in human cells. Surprisingly, these results demonstrated XRCC5 localization to PNA triplex-forming sites in the genome, suggesting the presence of a double-strand break intermediate. These results describe a powerful comparative approach for identifying structure-specific nucleic acid interactions and expand our understanding of the mechanisms of triplex structure recognition and repair.
KW - DNA binding protein
KW - DNA repair
KW - DNA structure
KW - gene therapy
KW - genome editing
KW - high-throughput screening
KW - protein–DNA interaction
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U2 - 10.1016/j.jbc.2022.102398
DO - 10.1016/j.jbc.2022.102398
M3 - Article
C2 - 35988651
AN - SCOPUS:85138214748
SN - 0021-9258
VL - 298
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 10
M1 - 102398
ER -