The three-dimensional organization of the genome supports regulated gene expression, recombination, DNA repair, and chromosome segregation during mitosis. Chromosome conformation capture (Hi-C) 1 , 2 has revealed a complex genomic landscape of internal chromosome structures in vertebrate cells 3 – 7 yet how sister chromatids topologically interact in replicated chromosomes has remained elusive due to their identical sequences. Here, we present sister-chromatid-sensitive Hi-C (scsHi-C) based on nascent DNA labeling with 4-thio-thymidine and nucleoside conversion chemistry. Genome-wide conformation maps of human chromosomes revealed that sister chromatid pairs interact most frequently at the boundaries of topologically associating domains (TADs). Continuous loading of a dynamic cohesin pool separates sister-chromatid pairs inside TADs and is required to focus sister chromatid contacts at TAD boundaries. We identified a subset of TADs that are overall highly paired, characterized by facultative heterochromatin, as well as insulated topological domains that form separately within individual sister chromatids. The rich pattern of sister chromatid topologies and our scsHi-C technology will make it possible to dissect how physical interactions between identical DNA molecules contribute to DNA repair, gene expression, chromosome segregation, and potentially other biological processes.

Temporal information about cellular RNA populations is essential to understand the functional roles of RNA. We have developed the hydrazine/NH4Cl/OsO4‐based conversion of 6‐thioguanosine (6sG) into A′, where A′ constitutes a 6‐hydrazino purine derivative. A′ retains the Watson–Crick base‐pair mode and is efficiently decoded as adenosine in primer extension assays and in RNA sequencing. Because 6sG is applicable to metabolic labeling of freshly synthesized RNA and because the conversion chemistry is fully compatible with the conversion of the frequently used metabolic label 4‐thiouridine (4sU) into C, the combination of both modified nucleosides in dual‐labeling setups enables high accuracy measurements of RNA decay. This approach, termed TUC‐seq DUAL, uses the two modified nucleosides in subsequent pulses and their simultaneous detection, enabling mRNA‐lifetime evaluation with unprecedented precision.

Temporal information about cellular RNAp opulations is essential to understand the functional roles of RNA. We have developed the hydrazine/NH 4 Cl/OsO 4 -based conversion of 6-thioguanosine (6sG) into A',w here A' constitutes a6hydrazino purine derivative.A' retains the Watson-Crickbasepair mode and is efficiently decoded as adenosine in primer extension assays and in RNAs equencing.B ecause 6sG is applicable to metabolic labeling of freshly synthesized RNA and because the conversion chemistry is fully compatible with the conversion of the frequently used metabolic label 4thiouridine (4sU) into C, the combination of both modified nucleosides in dual-labeling setups enables high accuracy measurements of RNAdecay. This approach, termed TUC-seq DUAL, uses the two modified nucleosides in subsequent pulses and their simultaneous detection, enabling mRNA-lifetime evaluation with unprecedented precision.

For this study, we utilized class-I and class-II preQ1-sensing riboswitches as model systems to decipher the structure-activity relationship of rationally designed ligand derivatives in vitro and in vivo. We found that synthetic preQ1 ligands with amino-modified side chains that protrude from the ligand-encapsulating binding pocket, and thereby potentially interact with the phosphate backbone in their protonated form, retain or even increase binding affinity for the riboswitches in vitro. They, however, led to significantly lower riboswitch activities in a reporter system in vivo in E. coli. Importantly, when we substituted the amino- by azido-modified side chains, the cellular activities of the ligands were restored for the class-I conditional gene expression system and even improved for the class-II counterpart. Kinetic analysis of ligand binding in vitro revealed enhanced on-rates for amino-modified derivatives while they were attenuated for azido-modified variants. This shows that neither high affinities nor fast on-rates are necessarily translated into efficient cellular activities. Taken together, our comprehensive study interconnects in vitro kinetics and in vitro thermodynamics of RNA-ligand binding with the ligands’ in vivo performance and thereby encourages azido- rather than amino-functionalized design for enhanced cellular activity.

The three-dimensional organization of the genome supports regulated gene expression, recombination, DNA repair, and chromosome segregation during mitosis. Chromosome conformation capture (Hi-C) 1-3 has revealed a complex genomic landscape of internal chromosome structures in vertebrate cells 4-11 yet how sister chromatids topologically interact in replicated chromosomes has remained elusive due to their identical sequences. Here, we present sister-chromatid-sensitive Hi-C (scsHi-C) based on nascent DNA labeling with 4-thio-thymidine. Genome-wide conformation maps of human chromosomes revealed that sister chromatid pairs interact most frequently at the boundaries of topologically associating domains (TADs). Continuous loading of a dynamic cohesin pool separates sister-chromatid pairs inside TADs and is required to focus sister chromatid contacts at TAD boundaries. We identified a subset of TADs that are overall highly paired, characterized by facultative heterochromatin, as well as insulated topological domains that form separately within individual sister chromatids. The rich pattern of sister chromatid topologies and our scsHi-C technology will make it possible to dissect how physical interactions between identical DNA molecules contribute to DNA repair, gene expression, chromosome segregation, and potentially other biological processes.

2-Aminopurine (Ap) is a fluorescent nucleobase analog that is frequently used as structure-sensitive reporter to study the chemical and biophysical properties of nucleic acids. In particular, thermodynamics and kinetics of RNA folding and RNAligand binding, as well as RNA catalytic activity are addressable by pursuing the Ap fluorescence signal in response to external stimuli. Site-specific incorporation of Ap into RNA is usually achieved by RNA solid-phase synthesis and requires appropriately functionalized Ap riboside building blocks. Here, we introduce a robust synthetic path toward a 2-aminopurine riboside phosphoramidite whose N 2 functionality is masked with the N-(di-n-butylamino)methylene group. This protection is considered advantageous over previously described N-(dimethylamino)methylene or acyl protection patterns needed for the fine-tuned deprotection conditions to achieve large synthetic RNAs.

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