Fu-Sen Liang

Engineering the ABA Plant Stress Pathway for Regulation of Induced Proximity

An abscisic acid–induced proximity system can be used in combination with other such systems to simultaneously control multiple cellular processes. Inducible Regulation with ABA Several systems exist to inducibly alter cellular behavior; however, they each have limitations. Turning to plants, Liang et al. determined that they could engineer systems with two protein components of the plant abscisic acid (ABA) pathway to create a chemically induced proximity system that responded to exogenously applied ABA (see the associated Perspective by Cutler). They demonstrated the ability of this ABA-induced proximity system to stimulate gene expression and control protein subcellular localization and activation of a signaling cascade in mammalian cells; furthermore, they showed that the ABA system could be combined with other induced proximity systems to control multiple proteins simultaneously. Studies with mice suggested that ABA was orally available and had a half-life suitable for analysis of biologic pathways. These characteristics of ABA, combined with its lack of toxicity, may make this system useful for applications in humans. Chemically induced proximity (CIP) systems use small molecules and engineered proteins to control and study biological processes. However, small molecule–based systems for controlling protein abundance or activities have been limited by toxicity, instability, cost, and slow clearance of the small molecules in vivo. To address these problems, we modified proteins of the plant abscisic acid (ABA) stress response pathway to control the proximity of cellular proteins and showed that the system could be used to regulate transcription, signal transduction, and subcellular localization of proteins in response to exogenously applied ABA. We also showed that the ABA CIP system can be combined with other CIP systems to simultaneously control multiple processes. We found that, when given to mice, ABA was orally available and had a 4-hour half-life. These properties, along with its lack of toxicity and low cost, suggest that ABA may be well suited for therapeutic applications and as an experimental tool to control diverse cellular activities in vivo.

Surface plasmon resonance study of RNA-aminoglycoside interactions

Publisher Summary An increasing number of small molecules with different structures have been discovered that bind to RNA. With the advanced techniques available to elucidate RNA structures and probe RNA–small molecule interactions, the underlying recognition principles regarding molecular details are just beginning to be revealed. For rational design of selective RNA binders, detailed knowledge of structure–affinity relationships between RNA and small molecules is required, as well as analysis of the binding specificity of various RNA sequences. Several methods have been used to investigate the binding between RNA and small molecules. Surface plasmon resonance (SPR) biosensor has been used to measure RNA–small molecule interactions and provides real-time monitoring without a labeling requirement. Although the SPR-based method has been applied to monitor macromolecular interactions, only more recently, with improved instrument detection accuracy, bindings between small molecules and macromolecules can be successfully monitored by SPR. This chapter focuses on describing procedures performed in the SPR binding assay, using aminoglycoside–bacterial ribosome 16S A-site binding as an example. The methods of data analysis to derive the equilibrium constant and binding stoichiometry are also discussed in the chapter.

Developmental biology: The early heart remodelled.

What factors direct the formation of heart muscle in the developing embryo? Unexpectedly, a chromatin-remodelling protein complex turns out to be a crucial determinant of cardiac-cell fate.

Constructing de Novo H2O2 Signaling via Induced Protein Proximity

A new chemical strategy has been developed to generate de novo signaling pathways that link a signaling molecule, H2O2, to different downstream cellular events in mammalian cells. This approach combines the reactivity-based H2O2 sensing with the chemically induced protein proximity technology. By chemically modifying abscisic acid with an H2O2-sensitive boronate ester probe, novel H2O2 signaling pathways can be engineered to induce transcription, protein translocation and membrane ruffle formation upon exogenous or endogenous H2O2 stimulation. This strategy has also been successfully applied to gibberellic acid, which provides the potential to build signaling networks based on orthogonal cell stimuli.

Evaluation of RNA-binding specificity of aminoglycosides with DNA microarrays

We have developed methods for using DNA array technology to probe the entire transcriptome to determine the RNA-binding specificity of ligands. Two methods were investigated. In the first method, the RNA-binding aminoglycoside antibiotic tobramycin was covalently linked to magnetic beads. The beads were bound to human liver mRNA and washed, and specifically bound RNA was eluted, amplified, and analyzed with DNA array technology. A small number of genes were found to bind specifically to the tobramycin beads. In the second method, the aminoglycoside ligand was added directly to the array hybridization reaction, and the signal was compared with a control experiment in the absence of ligand. The aminoglycosides were found to interfere with a small percentage of all hybridization events. These methods differ from traditional DNA array experiments in that the readout is a direct measure of the interaction between mRNA and a ligand, rather than an indirect measure of effect on expression. We expect that the results will lead to the discovery of new aminoglycoside-binding RNA motifs and may also have relevance toward understanding and overcoming the side effects observed with these antibiotics in the clinic.

Regulating miRNA-21 Biogenesis By Bifunctional Small Molecules

We report a new strategy to regulate microRNAs (miRNAs) biogenesis by using bifunctional small molecules that consist of a pre-miRNA binding unit connected by a linker to a Dicer inhibiting unit. In this effort, fluorescence polarization-based screening was used to identify neomycin as a pre-miR-21 binding ligand. Although neomycin cannot inhibit miR-21 maturation, linking it to the RNase inhibitor 1 forms the bifunctional conjugate 7A, which inhibits the production of miR-21. We expect that this strategy will be applicable to design other molecules for miRNA regulation.

Chemically Controlled Epigenome Editing through an Inducible dCas9 System

Although histone modifications are associated with gene activities, studies of their causal relationships have been difficult. For this purpose, we developed an inducible system integrating dCas9-based targeting and chemically induced proximity technologies to allow small molecule induced recruitment of P300 acetyltransferase and the acetylation of H3K27 at precise gene loci in cells. Employing the new technique, we elucidated the temporal order of histone acetylation and gene activation, as well as the stability of the installed histone modification.

Facile functionalization of FK506 for biological studies by the thiol–ene ‘click’ reaction

An efficient one-step functionalization of FK506 by the thiol–ene click (TEC) reaction is reported. This approach, which enables rapid and quantitative generation of bioactive FK1012 and FK506 derivatives, should facilitate biomedical applications of FK506-coupled molecules and expand the scope of the TEC reaction in natural product semi-synthesis.

Synthesis of ent-herbasolide

ent-Herbasolide is synthesised by elaboration of (+)-10-camphorsulfonyl chloride which involves homologation at C-10, cleavage of the C-2/C-3 bond, oxidation at C-5 and chain extension at C-3 of the camphor skeleton.

Design, synthesis and activity of light deactivatable microRNA inhibitor

miRNAs are key cellular regulators and their dysregulation is associated with many human diseases. They are usually produced locally in a spatiotemporally controlled manner to target mRNAs and regulate gene expression. Thus, developing chemical tools for manipulating miRNA with spatiotemporal precise is critical for studying miRNA. Herein, we designed a strategy to control miRNA biogenesis with light controllable inhibitor targeting the pre-miRNA processing by Dicer. By conjugating two non-inhibiting units, a low affinity Dicer inhibitor and a pre-miRNA binder, through a photocleavable linker, the bifunctional molecule obtained could inhibit miRNA production. Taking advantage of the photocleavable property of the linker, the bifunctional inhibitor can be fragmented into separate non-inhibiting units and therefore be deactivated by light. We expect that this strategy could be applied to generate chemical biological tools that allow light-mediated spatiotemporal control of miRNA maturation and contribute to the study of miRNA function.