Capturing Protein–Nucleic Acid Interactions by High‐Intensity Laser‐Induced Covalent Cross‐Linking^†

Abstract

Interactions of DNA with structural proteins such as histones, regulatory proteins and enzymes play a crucial role in major cellular processes such as transcription, replication and repair. The in vivo mapping and characterization of the binding sites of the involved biomolecules are of primary importance for a better understanding of genomic deployment that is implicated in tissue and developmental stagespecific gene expression regulation. The most powerful and commonly used approach to date is immunoprecipitation of chemically cross-linked chromatin (XChIP) coupled with sequencing analysis (ChIP-seq). While the resolution and the sensitivity of the high-throughput sequencing techniques have been constantly improved, little progress has been achieved in the cross-linking step. Because of its low efficiency, the use of the conventional UVC lamps remains very limited while the formaldehyde method was established as the "gold standard" cross-linking agent. Efficient biphotonic cross-linking of directly interacting nucleic acid-protein complexes by a single short UV laser pulse has been introduced as an innovative technique for overcoming limitations of conventionally used chemical and photochemical approaches. In this survey, the main available methods including the laser approach are critically reviewed for their ability to generate DNA-protein cross-links in vitro model systems and cells.

A Novel Adductomics Workflow Incorporating FeatureHunter Software: Rapid Detection of Nucleic Acid Modifications for Studying the Exposome

“…As shown in Figure 6, FeatureHunter will facilitate the proposal of a "top-down" NA adductomics approach, 29 by which patterns of adducts may be used to trace, and identify the originating exposure source, showing great promise as a valuable tool for unraveling the complexities of the exposome. FeatureHunter not only screens for user-defined features (i.e., NLs, product ions, and pair-peaks) in MS 1 and MS 2 spectra in input raw data files and builds extracted ion chromatograms (EICs) for precursors of interest, but also classifies and characterizes NA adducts. FeatureHunter brings a number of breakthrough innovations for postdata processing:…”

“…In this way, the more tags that are applied, the more accurate the classification of NA adducts by FeatureHunter. Third, FeatureHunter allows for an automated detection of "pair−peaks" with a fixed delta mass in MS 1 . SILMS is a powerful technique and has been frequently adopted in metabolomics 58 and DNA adductomics.…”

“…Using FeatureHunter, we further demonstrated, for the first time, novel types of rN adducts and RRCL (Figure 4B and 4E). Among these NA modifications, some were already present in the mixture of DNA, RNA, and BSA prior to formaldehyde treatment, but we determined with high confidence that others were directly induced by formaldehyde treatment, based on the observation of 12 C,d 0 -13 C,d 2 paired peaks (with a fixed mass difference of 3.015909, peak intensity ratio of 1.0 ± 0.3 and RT difference within 0.1 min) in MS 1 by FeatureHunter. The formaldehydeinduced NA modifications with their paired peaks are listed in Table S7.…”

“…Dynamic exclusion was enabled to prevent repeated acquisition of spectra from the most abundant ions during the analysis (exclude after one time triggered; exclusion duration: 6 s). To improve the sensitivity, an exclusion list was applied in MS 1 precursors selected for MS/MS fragmentation, including the unmodified 2′-dNs, rN, nB, the corresponding dimers and ion adducts (e.g., [2M + H] + , [M + Na] + , [M + K] + , and [M + NH 4 ] + ), as well as the background ions from solvent blank, as recommended previously. 39 2.5.…”

“…Additionally, FeatureHunter facilitates the detection of peak pairs with a fixed mass difference in MS 1 and effectively excludes false positive results arising from in-source fragmentation. The reliability and performance of FeatureHunter for NA adductomics were demonstrated through the analysis of: (i) 92 modified NA standards, (ii) NA modifications induced by formaldehyde and chlorambucil (CLB, an alkylating agent) in a mixture of calf thymus DNA (CT-DNA), yeast RNA, and protein (bovine serum albumin, BSA), and (iii) NA modifications present in the pooled urines of smokers and nonsmokers.…”