Genome-wide Identification of Structure-Forming Repeats as Principal Sites of Fork Collapse upon ATR Inhibition.

DNA polymerase stalling activates the ATR checkpoint kinase, which in turn suppresses fork collapse and breakage. Herein, we describe use of ATR inhibition (ATRi) as a means to identify genomic sites of problematic DNA replication in murine and human cells. Over 500 high-resolution ATR-dependent sites were ascertained using two distinct methods: replication protein A (RPA)-chromatin immunoprecipitation (ChIP) and breaks identified by TdT labeling (BrITL). The genomic feature most strongly associated with ATR dependence was repetitive DNA that exhibited high structure-forming potential. Repeats most reliant on ATR for stability included structure-forming microsatellites, inverted retroelement repeats, and quasi-palindromic AT-rich repeats. Notably, these distinct categories of repeats differed in the structures they formed and their ability to stimulate RPA accumulation and breakage, implying that the causes and character of replication fork collapse under ATR inhibition can vary in a DNA-structure-specific manner. Collectively, these studies identify key sources of endogenous replication stress that rely on ATR for stability.

Crystal Structure of an i-Motif from the HRAS Oncogene Promoter.

An i-motif is a non-canonical DNA structure implicated in gene regulation and linked to cancers. The C-rich strand of the HRAS oncogene, 5'-CGCCCGTGCCCTGCGCCCGCAACCCGA-3' (herein referred to as iHRAS), forms an i-motif in vitro but its exact structure was unknown. HRAS is a member of the RAS proto-oncogene family. About 19 % of US cancer patients carry mutations in RAS genes. We solved the structure of iHRAS at 1.77 Šresolution. The structure reveals that iHRAS folds into a double hairpin. The two double hairpins associate in an antiparallel fashion, forming an i-motif dimer capped by two loops on each end and linked by a connecting region. Six C-C+ base pairs form each i-motif core, and the core regions are extended by a G-G base pair and a cytosine stacking. Extensive canonical and non-canonical base pairing and stacking stabilizes the connecting region and loops. The iHRAS structure is the first atomic resolution structure of an i-motif from a human oncogene. This structure sheds light on i-motifs folding and function in the cell.

Machine Learning Informs RNA-Binding Chemical Space.

Small molecule targeting of RNA has emerged as a new frontier in medicinal chemistry, but compared to the protein targeting literature our understanding of chemical matter that binds to RNA is limited. In this study, we reported Repository Of BInders to Nucleic acids (ROBIN), a new library of nucleic acid binders identified by small molecule microarray (SMM) screening. The complete results of 36 individual nucleic acid SMM screens against a library of 24 572 small molecules were reported (including a total of 1 627 072 interactions assayed). A set of 2 003 RNA-binding small molecules was identified, representing the largest fully public, experimentally derived library of its kind to date. Machine learning was used to develop highly predictive and interpretable models to characterize RNA-binding molecules. This work demonstrates that machine learning algorithms applied to experimentally derived sets of RNA binders are a powerful method to inform RNA-targeted chemical space.

Three-Color Imaging Enables Simultaneous Screening of Multiple RNA Targets on Small Molecule Microarrays.

Small molecule microarray (SMM) technology has become a powerful tool used in high-throughput screening for target-based drug discovery. One area in which SMMs have found use is the identification of small molecule ligands for RNA. RNAs with unique secondary or tertiary three-dimensional structures are considered to be attractive targets for small molecules. Complex RNA structures can form hydrophobic pockets suitable for small molecule binding, representing an opportunity for developing novel therapeutics. Our lab has previously taken a target-based approach, screening a single target against many small molecules on an SMM platform. Here, we report a screening protocol for SMMs to investigate multiple RNAs simultaneously using multi-color imaging. By introducing a mixture containing different fluorophore-labeled RNAs, the fluorescence signal of each binding event can be observed simultaneously. Thus, the specificity of a hit compound binding to one RNA target over other highly abundant RNAs (such as tRNA or rRNA) can be easily evaluated. © 2020 Wiley Periodicals LLC. Basic Protocol: RNA screening on SMMs by multi-color imaging Support Protocol 1: Preparation of SMM slides Support Protocol 2: Fluorophore labeling of RNA through maleimide chemistry.