Methods in enzymology最新文献

RNAs are central mediators of genetic information flow and gene regulation that underlie diverse cell types and cell states across species. Thus, methods that can sense and respond to RNA profiles in living cells will have broad applications in biology and medicine. CellREADR - Cell access through RNA sensing by Endogenous ADAR (adenosine deaminase acting on RNA), is a programmable RNA sensor-actuator technology that couples the detection of a cell-defining RNA to the translation of an effector protein to monitor and manipulate the cell. The CellREADR RNA device consists of a 5' sensor region complementary to a cellular RNA and a 3' protein payload coding region. Payload translation is gated by the removal of a STOP codon in the sensor region upon base pairing with the cognate cellular RNA through an ADAR-mediated A-to-I editing mechanism ubiquitous to metazoan cells. CellREADR thus represents a new generation of programmable RNA device for monitoring and manipulating animal cells in ways that are simple, versatile, and generalizable across tissues and species. Here, we describe a detailed procedure for implementing CellREADR experiments in cell culture systems and in animals. The procedure includes sensor and payload design, cloning, validation and characterization in mammalian cell cultures. The in vivo protocol focuses on AAV-based delivery of CellREADR through expression vectors using brain tissue as an example. We describe current best practices and various experimental controls.

tRNA fragments (tRFs) are generated by cellular endogenous ribonuclease cleavage and play important roles in cellular processes and diseases states. Many questions regarding tRF functions remain to be studied and understood. Common sequencing techniques measure tRF after a size selection step that separates the full-length tRNA and tRF before sequencing library construction. The crucial information on the relationship of tRFs to their respective full-length tRNA in the same biological sample cannot be obtained in this way. We developed multiplex small RNA sequencing (MSR-seq) which measures the abundance as well as site-specific modification information on both full-length tRNA and their matching tRFs in the same sample. Here we describe the bioinformatic steps to obtain the tRF abundance data from the MSR-seq data using the publicly available pipeline in Github (https://github.com/Luke-F1875/MSRseq_data_processing_pipeline).

Cytidine-to-Uridine (C-to-U) RNA editing is a post-transcriptional modification essential for various biological processes. APOBEC deaminases mediate C-to-U editing which play critical role in cellular function and regulation. Advances in next-generation sequencing (NGS) technologies and analytical tools have provided powerful means to assess RNA editing activities and their physiological implications. However, inherent errors in NGS workflows-including reverse transcription, PCR amplification, and sequencing-complicate the detection of actual editing events. With error rates ranging from 10^-2 to 10^-3 per nucleotide, these technical artifacts can obscure APOBEC-mediated editing events occurring at similar frequencies. To address these challenges, in this chapter, we describe two established and optimized RNA sequencing strategies explicitly designed to detect low-frequency RNA editing events accurately while distinguishing them from NGS-associated errors. These methods are termed "circular RNA Sequencing Assay" and "Safe-Sequencing System (SSS)" and enable the reliable identification of RNA editing events (and also somatic mutations) at or below typical error thresholds.

Most recombinant proteins are expressed in model host organisms like Escherichia coli. Meanwhile, a significant number of enzymes require complex activation or special cofactors not available from standard hosts. The betaproteobacteria Thauera and Aromatoleum allow access to some of these enzymes, following procedures described in this chapter. The methods described enable transformation and conjugation of vectors into these species as alternate gene expression systems which allow fundamental studies of complex recombinant proteins as well as their biotechnological application.

Alcohol oxidation is a pivotal reaction in synthetic chemistry. Classical procedures typically involve the use of hypervalent reagents and organic solvents that often lead to poor selectivity, overoxidation issues and low atom-economy processes. The implementation of biocatalytic methods for this reaction is particularly appealing and several enzyme types have shown to catalyze this process although equilibrium issues, cofactor and coenzyme recycling or the use of external mediators limit the uptake of biocatalytic oxidations in large scale. Alcohol oxidases catalyze the non-reversible aerobic oxidation of alcohols to the corresponding carbonyls and have the potential to overcome current limitations in biocatalytic oxidations. In this chapter, we describe different protocols to find, obtain and characterize novel bacterial alcohol oxidases for synthetic chemistry.

In recent years, several PET-degrading enzymes have been identified from both known microorganisms and metagenomic sources in response to the growing environmental issue of polyethylene terephthalate (PET) accumulation. Despite this progress, there is a limited number of (ultra)high-throughput screening methods for assessing PET-hydrolyzing activity without relying on surrogate substrates. This method utilizes the coupled activity of ketoreductases (KREDs) and diaphorase to produce a fluorescent compound (resorufin) in the presence of PET degradation products, offering a more direct and efficient screening approach. A metagenomic KRED was coupled with the diaphorase from Clostridium kluyveri to enable the detection of the hydrolysis of PET degradation products catalyzed by the Bacillus subtilis BS2 esterase. The coupled reaction was established in water-in-oil microdroplets, encapsulating a single E. coli cell per droplet, demonstrating its potential for use in the ultrahigh-throughput screening of metagenomic libraries or randomized libraries for directed evolution campaigns.

Activation-induced cytidine deaminase (AID) and apolipoprotein B-mRNA editing catalytic polypeptide 3 (APOBEC3 or A3) proteins belong to the AID/APOBEC family of cytidine deaminases. While AID mediates somatic hypermutation and class-switch recombination in adaptive immunity, A3s restrict viruses and retroelements by hypermutation. Mis-regulated expression and off-target activity of AID/A3 can cause genome-wide mutations promoting oncogenesis, immune evasion, and therapeutic resistance due to tumor and viral evolution. In these contexts, inhibition of AID/A3 represents a promising therapeutic approach. Competitive inhibition could be achieved with different strategies: one class would be small molecules that bind in the catalytic pocket (active site) and block access for the substrate cytidine. Another type of larger molecule inhibitor would bind the enzymes' surface more broadly and compete with the binding of the polynucleotide substrates prior to deamination catalysis. Several biochemical assays developed to assess AID/A3 activity can be employed to screen for potential inhibitors. These include in cellulo and in vitro activity-based as well as binding-based assays. In this chapter, we discuss the key considerations for designing robust enzyme assays and provide an overview of assays that we and others have established or modified for specific applications in AID/A3 enzymology, including measurement of inhibition. We provide detailed protocols for the two most widely used in vitro enzyme assays that directly measure the activities of purified AID/A3s on DNA and/or RNA substrates, namely, the gel-based alkaline cleavage assay and multiple variations of PCR/sequencing-based assays.

Mammalian polyamines, namely putrescine, spermidine, and spermine, have been implicated in many cellular homeostatic processes. Polyamines play a critical role in skeletal health as evidenced by recent studies and by skeletal disorders caused by polyamine imbalances, such as Snyder-Robinson Syndrome (SRS). However, very little is still known about the role of polyamines within bone development, homeostasis, and metabolism. Human bone marrow derived mesenchymal stromal cells (MSCs) provide a unique opportunity to study polyamines at a cellular and molecular level within the context of osteogenic differentiation and calcium deposition. Through in vitro work, mechanistic understanding of the role of polyamines within osteogenesis as well as the consequences of polyamine imbalance can provide new insights into potential therapeutics for those experiencing polyaminopathies. This chapter describes procedures to develop a human primary cell culture system and quantify osteoblastogenesis as a function of polyamine modulation.

Combination therapies which target both polyamine biosynthesis and polyamine transport have shown promise as anti-cancer strategies and as potentiators of the immune response. While polyamine biosynthesis inhibitors like difluoromethylornithine (DFMO) exist, cancers often escape via upregulated polyamine import. As a result, polyamine transport inhibitors (PTIs) are needed to inhibit polyamine uptake and create a 'full-court press' on polyamine metabolism. As new PTIs are developed, they need to be ranked for their ability to inhibit polyamine uptake. This paper describes three polyamine transport assays to evaluate polyamine transport inhibition. The first tests the ability of the PTI to inhibit the uptake of an anthracene-containing polyamine poison (Ant44). The second assay evaluates the ability of the PTI to inhibit the uptake of a rescuing dose of spermidine into DFMO-treated cells. The final assay is the gold standard for the field and involves determining the concentration of PTI needed to inhibit 50 % of the uptake of each of the radiolabeled native polyamines: ³H-putrescine, ³H-spermidine or ¹⁴C-spermine. These assays provide EC₅₀ and IC₅₀ values which allow a formal ranking of transport inhibition potency to aid in PTI selection.

Polyamines (PAs) are small polycationic alkylamines that are essential for numerous cellular processes and found in all living cells. The three principal polyamines, putrescine (PUT), spermidine (SPD), and spermine (SPM), have been shown to play crucial roles in cellular function and implicated in several diseases including infectious diseases, cancer and neurodegenerative disorders. As such, the enzymes involved in polyamine biosynthesis are promising targets for developing antimicrobial, antineoplastic and neuroprotective therapies. Aminopropyl transferases (APTs) are key enzymes in this pathway, catalyzing the formation of spermidine from putrescine and spermine from spermidine. While in most eukaryotes and prokaryotes, the spermidine synthase and spermine synthase activities are catalyzed by distinct enzymes, some organisms such as Plasmodium falciparum have a single enzyme, which catalyzes both reactions with varying efficiency. To date, efforts to inhibit APTs have focused primarily on substrate analogs, often with limited selectivity. A major challenge in discovering novel inhibitors has been the lack of an assay suitable for high-throughput chemical screening. We have recently developed DAB-APT, the first fluorescence-based assay for measuring APT activity, using 1,2-diacetyl benzene (DAB) which reacts with putrescine, spermidine, and spermine to form fluorescent conjugates, with fluorescence intensity correlating to carbon chain length. The DAB-APT assay has been validated using APT enzymes from Saccharomyces cerevisiae, and P. falciparum, and has been found to be suitable for high-throughput screening of large chemical libraries. This assay represents a significant advancement, offering a valuable tool for identifying potential inhibitors of APT enzymes and accelerating drug discovery efforts in cancer, neurobiology, and infectious diseases.