Methods in enzymology最新文献

Natural products are a fascinating source of chemical diversity and their biosynthetic pathways of biological complexity. The investigation and engineering of biosynthetic pathways towards polyketides in Actinomycetes provides challenges across all steps of the mutagenesis procedure. The typically GC-rich and long genes require robust PCR protocols. The resulting amplicons, often exceeding 10 kbp in length, require equally robust cloning procedures. Finally, the genetic manipulation of Actinomycetes, especially Streptomyces spp., calls for specialized procedures, in particular when the construction of several hundred variants is needed. This chapter will detail methods for all three steps of the process and have been previously used to generate numerous polyketide synthase variants in several Actinomycete species.

Although RNA-seq data are becoming more widely available for biomedical research, most datasets for short non-coding RNAs (sncRNAs) primarily focus on microRNA analysis using standard RNA-seq, which captures only sncRNAs with 5'-phosphate (5'-P) and 3'-hydroxyl (3'-OH) ends. Standard RNA-seq fails to sequence sncRNAs with different terminal phosphate states, including tRNA halves, the most abundant class of tRNA-derived sncRNAs that play diverse roles in various biological processes. tRNA halves are produced through the endoribonucleolytic cleavage of mature tRNA anticodon loops. The responsible endoribonucleases, such as Angiogenin, commonly leave a 2',3'-cyclic phosphate (cP) at the 3'-end of 5'-tRNA halves and forms a 5'-OH end of 3'-tRNA halves, making them incompatible with standard RNA-seq. We developed a method named "cP-RNA-seq" that selectively amplifies and sequences tRNA halves and other cP-containing sncRNAs. Here we describe a detailed and recently updated cP-RNA-seq protocol. In this method, the 3'-end of all sncRNAs, except those containing a cP, are cleaved through periodate treatment after phosphatase treatment. Consequently, adaptor ligation and cDNA amplification steps are exclusively applied to cP-containing sncRNAs. Our cP-RNA-seq only requires commercially available reagents and is broadly applicable for the global identification of tRNA halves and other cP-containing sncRNA repertoires in various transcriptomes.

Angiogenin (RNase 5) is an unusual member of the RNase A family with very weak RNase activity and a preference for tRNA. The tRNAs cleaved by angiogenin are thought to have a variety of roles in cellular processes including translation reprogramming, apoptosis, angiogenesis, and neuroprotection. We recently demonstrated that angiogenin is potently activated by the cytoplasmic 80S ribosome. Angiogenin's binding to the ribosome rearranges the C-terminus of the protein, opening the active site for the cleavage of tRNA delivered to the ribosomal A site which angiogenin occupies. Here, we describe the biochemical procedure to test angiogenin's activation by the ribosome using the assay termed the Ribosome Stimulated Angiogenin Nuclease Assay (RiSANA). RiSANA can be used to test the activity of wild-type or mutant angiogenin, or other RNases, against different tRNAs and with different ribosome complexes. For example, given that angiogenin has been implicated in anti-microbial activity, we tested the ability of bacterial 70S ribosomes to stimulate angiogenin activity and found that the E. coli ribosome does not stimulate angiogenin. We also assayed whether angiogenin's closest homolog, RNase 4, could be stimulated by the ribosome, but unlike angiogenin this enzyme was not further activated by the ribosome. The RiSANA assay promises to reveal new aspects of angiogenin mechanism and may aid in the development of new diagnostic tools and therapeutics.

Base editing and other precision editing agents have transformed the utility and therapeutic potential of CRISPR-based genome editing. While some native enzymes edit efficiently with their nature-derived function, many enzymes require rational engineering or directed evolution to enhance the compatibility with mammalian cell genome editing. While many methods of engineering and directed evolution exist, plate-based discrete evolution offers an ideal balance between ease of use and engineering power. Here, we describe a detailed method for the bacterial directed evolution of CRISPR base editors that compounds technical ease with flexibility of application.

Adenosine-to-Inosine (A-to-I) RNA editing is the most prevalent type of RNA editing, in which adenosine within a completely or largely double-stranded RNA (dsRNA) is converted to inosine by deamination. RNA editing was shown to be involved in many neurological diseases and cancer; therefore, detection of A-to-I RNA editing and quantitation of editing levels are necessary for both basic and clinical biomedical research. While high-throughput sequencing (HTS) is widely used for global detection of editing events, Sanger sequencing is the method of choice for precise characterization of editing site clusters (hyper-editing) and for comparing levels of editing at a particular site under different environmental conditions, developmental stages, genetic backgrounds, or disease states. To detect A-to-I editing events and quantify them using Sanger sequencing, RNA samples are reverse transcribed, cDNA is amplified using gene-specific primers, and then sequenced. The chromatogram outputs are then compared to the genomic DNA sequence. As editing occurs in the context of dsRNA, the reverse transcription step is performed at a temperature as high as 65 °C, using thermostable reverse transcriptase to open double-stranded structures. However, this measure alone is insufficient for transcripts possessing long stems comprised of hundreds of nucleotide pairs. Consequently, the editing levels detected by Sanger sequencing are significantly lower than those obtained by HTS, and the amplification yield is low. We suggest that the reverse transcription is biased towards unedited transcripts, and the severity of the bias is dependent on the transcript's secondary structure. Here, we show how this bias can be significantly reduced to allow reliable detection of editing levels and sufficient product yield.

Group II introns are transposable elements that can propagate in host genomes through the "copy and paste" mechanism. They usually comprise RNA and protein components for effective propagation. Recently, we found that some bacterial GII-C introns without protein components had multiple copies in their resident genomes, implicating their potential transposition activity. We demonstrated that some of these systems are active for hydrolytic DNA cleavage and proved their DNA manipulation capability in bacterial or mammalian cells. These introns are therefore named HYdrolytic Endonucleolytic Ribozymes (HYERs). Here, we provide a detailed protocol for the systematic identification and characterization of HYERs and present our perspectives on its potential application in nucleic acid manipulation.

Selecting the appropriate mode of biocatalysis application is crucial for optimizing efficiency and sustainability. This chapter provides a comprehensive guide on key metrics to describe biocatalyst performance, including kinetic parameters such as reaction rates, cofactor requirements, dissociation constants (K_D), maximum velocities (V_max), turnover numbers (k_cat), and Michaelis constants (K_M). Additionally, it discusses biocatalysis metrics like turnover frequency (TOF), environmental factors (E-Factor), atom economy, productivities, and Life Cycle Assessment (LCA). The chapter also explores application types, focusing on whole-cell and cell-free enzyme applications, and offers a practical guide on selecting the most suitable mode of application based on specific project requirements. By integrating these considerations, researchers can effectively harness biocatalysis for innovative and sustainable solutions in various industrial processes.

Polyamines, spermidine (Spd) and Spermine (Spm), are polycations that serve a number of important biological functions. The tissue contents of polyamines are tightly regulated through their cellular import and export, as well as their metabolism (anabolism and catabolism). Polyamine catabolism in mediated via the spermidine/spermine N1-acetyltransferase (SAT1)/acetylpolyamine oxidase (APOX) cascade and oxidation of Spm by spermine oxidase (SMOX). The expression of SAT1 and SMOX increases in injured organs in response to trauma, ischemia/reperfusion, sepsis, and exposure to toxic compounds. Cisplatin is a highly effective chemotherapeutic agent that is used for the treatment of a variety of solid tumors. Its anti-tumor activity is mediated via its ability to form stable DNA adducts that inhibit the growth of actively proliferating cells. However, cisplatin also can lead to severe off-target deleterious effects (e.g., nephrotoxicity and ototoxicity), and because of such adverse effects the use of cisplatin has to be discontinued in many patients. Understanding and decoupling the therapeutic and toxic effects of cisplatin will lead to more effective use of this and other platinum-derived compounds in the treatment of cancer patients. Acute and chronic exposure to cisplatin in mice leads to severe renal tubular injuries and an increase in the expression of SAT1 and SMOX while the ablation of their genes in mice reduces the severity of nephrotoxic injuries caused by cisplatin. Furthermore, neutralization of the toxic by-products of polyamine degradation reduce the severity if cisplatin nephrotoxicity. These observations suggest that interventions targeting the adverse effects of enhanced polyamine catabolism may provide effective therapies by reducing the toxic effects of cisplatin without affecting its anti-neoplastic activity.

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.