Methods in enzymology最新文献

RNA plays key roles not only as an intermediate between DNA and protein during translation, but also a functional biocatalyst for gene regulation, cell development, environmental interactions and various diseases. In addition to the classical four nucleotides, many chemical modifications located in different positions of the nucleotide further affect and diversify RNA structures and functions. Synthetic RNAs containing these chemical modifications, which are usually made through the well-developed solid phase synthesis, are important toolsets to study RNA biology and develop new therapeutics. This chapter, taking the synthesis of RNAs containing m³C, m⁴C, and m⁴₂C modifications as examples, summarizes the experimental protocols from the synthesis of single nucleoside & phosphoramidite building blocks to the preparation of RNA oligonucleotides using a solid-phase synthesizer, as well as their biophysical and biochemical characterizations, providing a general template for investigating other modified RNAs.

Exactly two decades ago, the ability to use high-throughput RNA sequencing technology to identify sites of editing by ADARs was employed for the first time. Since that time, RNA sequencing has become a standard tool for researchers studying RNA biology and led to the discovery of RNA editing sites present in a multitude of organisms, across tissue types, and in disease. However, transcriptome-wide sequencing is not without limitations. Most notably, RNA sequencing depth of a given transcript is correlated with expression, and sequencing depth impacts the ability to robustly detect RNA editing events. This chapter focuses on a method for enrichment of low-abundance transcripts that can facilitate more efficient sequencing and detection of RNA editing events. An important note is that while we describe aspects of the protocol important for capturing intron-containing transcripts, this probe-based enrichment method could be easily modified to assess editing within any low-abundance transcript. We also provide some perspectives on the current limitations as well as important future directions for expanding this technology to gain more insights into how RNA editing can impact transcript diversity.

Assaying enzymes and microbes for activity for degradation of polymeric lignin is inherently challenging to do. This article describes several methods that our research group has developed for assay of lignin-oxidising enzymes and lignin-degrading microbes. The assay methods involve (1) colorimetric assays involving chemically nitrated lignin; (2) changes in molecular weight using gel filtration chromatography; (3) delignification of lignocellulose using Klason assay; (4) colorimetric assays for release of low molecular weight phenols and carbonyl compounds.

The broad substrate specificity of laccases makes these enzymes suitable for a wide range of applications. The use of protein engineering strategies to modulate the catalytic properties of these enzymes is a promising strategy to expand their use in the sustainable economy. Here we describe the construction of laccase-xylanase bifunctional enzyme by insertional fusion using a procedure based on the rational design starting with the analysis of the 3D-structure of laccase to select positions for the insertion of the xylanase domain, followed by the creation of the fusion construct by ligation of overlapping fragments generated by PCR. Finally, the heterologous expression and biochemical characterization of the laccase and xylanase activities of the fusion protein is described and demonstrate significant increase in the laccase activity. These protocols can be applied to the fusion of any pair of proteins.

Natural products are a rich source of bioactive compounds, which are encoded by the biosynthetic gene clusters (BGCs). Genome mining is an essential strategy for identifying and characterizing BGCs. Targeted genome mining excels in the identification of similar BGCs based on key biosynthetic enzymes across multiple genomes. This chapter details the use of both manual and automated targeted genome mining to identify members of the FK-family BGCs (rapamycin, FK520/506). We describe the process of selecting query proteins, evaluating genomic context, and determining the presence of putative BGCs. Additionally, to streamline the manual process, we used GATOR-GC, a computational tool that identifies similar BGCs using required and optional proteins, performs comparative genomic analysis, deduplicates redundant BGCs, and generates visualizations of gene conservation and BGC diversity. Applying this approach, we showed how to identify FK-family members, both by looking into the cluster conservation diagrams, and the clustered heatmap summarizing all-vs-all BGC comparisons. The methods outlined here can be adapted for mining other natural product families, offering a scalable framework for uncovering novel biosynthetic pathways. Beyond natural product discovery, GATOR-GC provides broader applications for analyzing gene cluster conservation, organization, and evolutionary patterns.

Emerging nascent polypeptides from ribosomes and their protein N-termini have significant impact on protein stability, folding, interaction and subcellular targeting. To profile elongating nascent polypeptides on a proteome-wide scale, here we present a streamlined protocol that combined a biochemical enrichment of puromycin-labeled nascent polypeptides and mass spectrometry-based quantitative proteomics. This chapter includes the detailed protocol for metabolic pulse labeling with puromycin and SILAC amino acids, immunoprecipitation for nascent polypeptides, fractionation and enrichment of N-terminal acetylated peptides as well as MS and data analysis. These protocols are illustrated using HeLa cells treated with cycloheximide, a protein synthesis inhibitor, but can be broadly applied to any cell culture systems, including primary cultures, or any treatments (e.g., drugs).

Artificial metalloenzyme (ArM) design is an attractive approach for deciphering the functional determinants of native enzymes or imparting new functions. Metalloproteins with redox cofactors catalyze critical reactions enabled by optimized primary, secondary, and outer-sphere interactions that facilitate efficient electron transfer. Controlling outer-sphere interactions to tune reactivity remains a challenge. Inspired by the common coordination motifs of copper (Cu) proteins, we have designed artificial Cu proteins (ArCuPs), extensively characterized them, and demonstrated their H₂O₂, O₂, and C-H oxidation reactivity to abiotic substrates. By selectively modifying outer-sphere solvent reorganization energy, we have shown that we can control C-H peroxidation activity. This chapter describes methods to design ArCuPs and their detailed characterization, including electrochemical C-H oxidation, and the procedures for determining reorganization energies using electrochemistry as a readily available laboratory tool.

APOBEC cytidine deaminases guard cells in a variety of organisms from invading viruses and foreign nucleic acids. Recently, several human APOBECs have been implicated in mutating evolving cancer genomes. Expression of APOBEC3A and APOBEC3B in yeast allowed experimental derivation of the substitution patterns they cause in dividing cells, which provided critical links to these enzymes in the etiology of the COSMIC single base substitution (SBS) signatures 2 and 13 in human tumors. Additionally, the ability to scale yeast experiments to high-throughput screens allows use of this system to also investigate cellular pathways impacting the frequency of APOBEC-induced mutation. Here, we present validated methods utilizing yeast to determine APOBEC mutation signatures, genetic interactors, and chromosomal substrate preferences. These methods can be employed to assess the potential of other human APOBECs and APOBEC orthologs in different species to contribute to cancer genome evolution as well as define the pathways that protect the nuclear genome from inadvertent APOBEC activity during viral restriction.