{"doi":"10.1101/2023.09.27.559819","title":"Studies of DNA ‘breathing’ by polarization-sweep single-molecule fluorescence microscopy of exciton-coupled (iCy3) <sub>2</sub> dimer-labeled DNA fork constructs","abstract":"Abstract Local fluctuations of the sugar-phosphate backbones and bases of DNA (often called DNA ‘breathing’) play a variety of critical roles in controlling the functional interactions of the DNA genome with the protein complexes that regulate it. Here we present a single-molecule fluorescence method that we have used to measure and characterize such conformational fluctuations at and near biologically important positions in model DNA replication fork constructs labeled with exciton-coupled cyanine [(iCy3) 2 ] dimer probes. Previous work has shown that the constructs that we test here exhibit a broad range of spectral properties at the ensemble level, and these differences can be structurally and dynamically interpreted using our present methodology at the single-molecule level. The (iCy3) 2 dimer has one symmetric (+) and one anti-symmetric (–) exciton with respective transition dipole moments oriented perpendicular to one another. We excite single molecule samples using a continuous-wave linearly polarized laser with polarization direction continuously rotated at the frequency 1 MHz. The ensuing fluorescence signal is modulated as the laser polarization alternately excites the symmetric and the anti-symmetric excitons of the (iCy3) 2 dimer probe. Phase-sensitive detection of the modulated signal provides information about the distribution of local conformations and conformational interconversion dynamics of the (iCy3) 2 probe. We find that at most construct positions that we examined the (iCy3) 2 dimer-labeled DNA fork constructs can adopt four topologically distinct conformational macrostates. These results suggest that in addition to observing DNA breathing at and near ss-dsDNA junctions, our new methodology should be useful to determine which of these pre-existing macrostates are recognized by, bind to, and are stabilized by various genome regulatory proteins.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2023,"id":400989,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9508,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":825658,"name":"Claire S. Albrecht","orcid":"0000-0003-3887-2683","position":1,"is_corresponding":false},{"id":1084878,"name":"Patrick J. Herbert","orcid":"0000-0003-2823-4514","position":2,"is_corresponding":false},{"id":831576,"name":"Dylan Heussman","orcid":null,"position":3,"is_corresponding":false},{"id":1085372,"name":"Anabel Chang","orcid":null,"position":4,"is_corresponding":false},{"id":364556,"name":"Peter H. von Hippel","orcid":"0000-0003-2512-8097","position":5,"is_corresponding":false},{"id":576807,"name":"Andrew H. Marcus","orcid":"0000-0003-2109-327X","position":6,"is_corresponding":false},{"id":1085371,"name":"Jack Maurer","orcid":null,"position":0,"is_corresponding":true}],"reference_count":38,"raw_metadata":null,"created_at":"2026-07-19T01:20:08.195423Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}