Chemical strategies for antisense antibiotics.

Antibacterial resistance is a severe threat to modern medicine and human health. To stay ahead of constantly-evolving bacteria we need to expand our arsenal of effective antibiotics. As such, antisense therapy is an attractive approach.

Catechol-Siderophore Mimics Convey Nucleic Acid Therapeutics into Bacteria.

Antibacterial resistance is a major threat for human health. There is a need for new antibacterials to stay ahead of constantly-evolving resistant bacteria. Nucleic acid therapeutics hold promise as powerful antibiotics, but issues with their delivery hamper their applicability.

Pervasive transcriptome interactions of protein-targeted drugs.

The off-target toxicity of drugs targeted to proteins imparts substantial health and economic costs. Proteome interaction studies can reveal off-target effects with unintended proteins; however, little attention has been paid to intracellular RNAs as potential off-targets that may contribute to toxicity. To begin to assess this, we developed a reactivity-based RNA profiling methodology and applied it to uncover transcriptome interactions of a set of Food and Drug Administration-approved small-molecule drugs in vivo.

Exploring antibiotic resistance with chemical tools.

Antibiotic resistance is an enormous problem that is accountable for over a million deaths annually, with numbers expected to significantly increase over the coming decades. Although some of the underlying causes leading up to antibiotic resistance are well understood, many of the molecular processes involved remain elusive. To better appreciate at a molecular level how resistance emerges, customized chemical biology tools can offer a solution.

A Quenched Size-Expanded Nucleotide Reports Activity of the Leukemia Biomarker Terminal Deoxynucleotidyl Transferase (TdT).

Terminal deoxynucleotidyl Transferase (TdT) is a template-independent DNA polymerase that plays an essential role in the human adaptive immune system and is upregulated in several types of leukemia. It has therefore gained interest as a leukemia biomarker and potential therapeutic target. Herein, we describe a FRET-quenched fluorogenic probe based on a size-expanded deoxyadenosine that reports directly on TdT enzymatic activity.

Chemical RNA Cross-Linking: Mechanisms, Computational Analysis, and Biological Applications.

In recent years, RNA has emerged as a multifaceted biomolecule that is involved in virtually every function of the cell and is critical for human health. This has led to a substantial increase in research efforts to uncover the many chemical and biological aspects of RNA and target RNA for therapeutic purposes. In particular, analysis of RNA structures and interactions in cells has been critical for understanding their diverse functions and druggability.

Affinity-Based Profiling of the Flavin Mononucleotide Riboswitch.

Riboswitches are structural RNA elements that control gene expression. These naturally occurring RNA sensors are of continued interest as antibiotic targets, molecular sensors, and functional elements of synthetic circuits. Here, we describe affinity-based profiling of the flavin mononucleotide (FMN) riboswitch to characterize ligand binding and structural folding.

Chemical reversible crosslinking enables measurement of RNA 3D distances and alternative conformations in cells.

Three-dimensional (3D) structures dictate the functions of RNA molecules in a wide variety of biological processes. However, direct determination of RNA 3D structures in vivo is difficult due to their large sizes, conformational heterogeneity, and dynamics. Here we present a method, Spatial 2'-Hydroxyl Acylation Reversible Crosslinking (SHARC), which uses chemical crosslinkers of defined lengths to measure distances between nucleotides in cellular RNA.

Optimized photochemistry enables efficient analysis of dynamic RNA structuromes and interactomes in genetic and infectious diseases.

Direct determination of RNA structures and interactions in living cells is critical for understanding their functions in normal physiology and disease states. Here, we present PARIS2, a dramatically improved method for RNA duplex determination in vivo with >4000-fold higher efficiency than previous methods. PARIS2 captures ribosome binding sites on mRNAs, reporting translation status on a transcriptome scale.

The chemistry and applications of RNA 2'-OH acylation.

RNA is a versatile biomolecule with a broad range of biological functions that go far beyond its initially described role as a simple information carrier. The development of chemical methods to control, manipulate and modify RNA has the potential to yield new insights into its many functions and properties. Traditionally, most of these methods involved the chemical modification of RNA structure using solid-state synthesis or enzymatic transformations.

Trapping Transient RNA Complexes by Chemically Reversible Acylation.

RNA-RNA interactions are essential for biology, but they can be difficult to study due to their transient nature. While crosslinking strategies can in principle be used to trap such interactions, virtually all existing strategies for crosslinking are poorly reversible, chemically modifying the RNA and hindering molecular analysis. We describe a soluble crosslinker design (BINARI) that reacts with RNA through acylation.

Polyacetate and Polycarbonate RNA: Acylating Reagents and Properties.

Acylation of RNA at 2'-OH groups is widely applied in mapping RNA structure and recently for controlling RNA function. Reactions are described that install the smallest 2-carbon acyl groups on RNA-namely, 2'-O-acetyl and 2'-O-carbonate groups. Hybridization and thermal melting experiments are performed to assess the effects of the acyl groups on duplex formation.

Water-Soluble Leaving Group Enables Hydrophobic Functionalization of RNA.

Attachment of hydrophobic groups to RNA is challenging because of their poor aqueous solubility. One-step acylation of RNA 2'-OH groups in water using a water-soluble imidazole leaving group is described. The effect of the hydrophobic groups on hybridization is reported.

RNA Control by Photoreversible Acylation.

External photocontrol over RNA function has emerged as a useful tool for studying nucleic acid biology. Most current methods rely on fully synthetic nucleic acids with photocaged nucleobases, limiting application to relatively short synthetic RNAs. Here we report a method to gain photocontrol over RNA by postsynthetic acylation of 2'-hydroxyls with photoprotecting groups.

Fingerprints of Modified RNA Bases from Deep Sequencing Profiles.

Posttranscriptional modifications of RNA bases are not only found in many noncoding RNAs but have also recently been identified in coding (messenger) RNAs as well. They require complex and laborious methods to locate, and many still lack methods for localized detection. Here we test the ability of next-generation sequencing (NGS) to detect and distinguish between ten modified bases in synthetic RNAs.

Fluorogenic Templated Reaction Cascades for RNA Detection.

Nucleic acids detection is essential to the study of biological processes and to diagnosis of pathological states. Although PCR is highly effective in vitro, methods that can function without prior sample preparation, thermal cycling, or enzymes are of interest due to their simplicity. Most current non-PCR detection methods rely on linear signal amplification, which hinders the detection of small amounts of genetic material.

DNA polymerase θ specializes in incorporating synthetic expanded-size (xDNA) nucleotides.

DNA polymerase θ (Polθ) is a unique A-family polymerase that is essential for alternative end-joining (alt-EJ) of double-strand breaks (DSBs) and performs translesion synthesis. Because Polθ is highly expressed in cancer cells, confers resistance to ionizing radiation and chemotherapy agents, and promotes the survival of homologous recombination (HR) deficient cells, it represents a promising new cancer drug target. As a result, identifying substrates that are selective for this enzyme is a priority.

Orthogonal photoswitching in a multifunctional molecular system.

The wavelength-selective, reversible photocontrol over various molecular processes in parallel remains an unsolved challenge. Overlapping ultraviolet-visible spectra of frequently employed photoswitches have prevented the development of orthogonally responsive systems, analogous to those that rely on wavelength-selective cleavage of photo-removable protecting groups. Here we report the orthogonal and reversible control of two distinct types of photoswitches in one solution, that is, a donor-acceptor Stenhouse adduct (DASA) and an azobenzene.

Ciprofloxacin-Photoswitch Conjugates: A Facile Strategy for Photopharmacology.

Photopharmacology aims to locally treat diseases and study biological processes with photoresponsive drugs. Herein, easy access to photoswitchable drugs is crucial, which is supported by simple and robust drug modifications. We investigated the possibility of creating drugs that can undergo remote activation and deactivation with light, by conjugating molecular photoswitches to the exterior of an existing drug in a single chemical step.

Light-Controlled Histone Deacetylase (HDAC) Inhibitors: Towards Photopharmacological Chemotherapy.

Cancer treatment suffers from limitations that have a major impact on the patient's quality of life and survival. In the case of chemotherapy, the systemic distribution of cytotoxic drugs reduces their efficacy and causes severe side effects due to nonselective toxicity. Photopharmacology allows a novel approach to address these problems because it employs external, local activation of chemotherapeutic agents by using light.

Wavelength-selective cleavage of photoprotecting groups: strategies and applications in dynamic systems.

Photocleavable protecting groups (PPGs) are extensively used in chemical and biological sciences. In their application, advantage is taken of using light as an external, non-invasive stimulus, which can be delivered with very high spatiotemporal precision. More recently, orthogonally addressing multiple PPGs, in a single system and with different wavelengths of light, has been explored.

Bacterial patterning controlled by light exposure.

Patterning of multiple bacterial strains in one system is achieved by employing a single photo-activated antibiotic. Varying the light-exposure time results in zones with mixed and single populations.

Proteasome inhibitors with photocontrolled activity.

Proteasome inhibitors are widely used in cancer treatment as chemotherapeutic agents. However, their employment often results in severe side effects, due to their non-specific cytotoxicity towards healthy tissue. This problem might be overcome by using a photopharmacological approach, that is, by attaining external, dynamic, spatiotemporal photocontrol over the activity of a cytotoxic agent, achieved by the introduction of a photoswitchable moiety into its molecular structure.

Orthogonal control of antibacterial activity with light.

Selection of a single bacterial strain out of a mixture of microorganisms is of crucial importance in healthcare and microbiology research. Novel approaches that can externally control bacterial selection are a valuable addition to the microbiology toolbox. In this proof-of-concept, two complementary antibiotics are protected with photocleavable groups that can be orthogonally addressed with different wavelengths of light.

Photocaging of carboxylic acids: a modular approach.

Photocaged compounds are important tools for studying and regulating multiple processes, including biological functions. Reported herein is the use of the Passerini multicomponent reaction for modular preparation of photocaged carboxylic acids. The reaction is compatible with several functionalities and proceeds smoothly both in water and dichloromethane.