Methods in enzymology最新文献

CellREADR: An ADAR-based RNA sensor-actuator device.

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-10 DOI: 10.1016/bs.mie.2024.11.027

Xiaolu Yang, Kehali Woldemichael, Xiao Guo, Shengli Zhao, Yongjun Qian, Z Josh Huang

{"title":"CellREADR: An ADAR-based RNA sensor-actuator device.","authors":"Xiaolu Yang, Kehali Woldemichael, Xiao Guo, Shengli Zhao, Yongjun Qian, Z Josh Huang","doi":"10.1016/bs.mie.2024.11.027","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.11.027","url":null,"abstract":"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.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"710 ","pages":"207-227"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143053003","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Bioinformatics of simultaneous, quantitative measurements of full-length tRNA and tRNA fragments by MSR sequencing.

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-11-22 DOI: 10.1016/bs.mie.2024.11.009

Luke R Frietze, Tao Pan

引用次数: 0

A probe-based capture enrichment method for detection of A-to-I editing in low abundance transcripts.

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-02 DOI: 10.1016/bs.mie.2024.11.033

Emma Lamb, Dyuti Pant, Boyoon Yang, Heather A Hundley

{"title":"A probe-based capture enrichment method for detection of A-to-I editing in low abundance transcripts.","authors":"Emma Lamb, Dyuti Pant, Boyoon Yang, Heather A Hundley","doi":"10.1016/bs.mie.2024.11.033","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.11.033","url":null,"abstract":"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.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"710 ","pages":"55-75"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143052974","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Obstacles in quantifying A-to-I RNA editing by Sanger sequencing.

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-02 DOI: 10.1016/bs.mie.2024.11.032

Alla Fishman, Ayelet T Lamm

{"title":"Obstacles in quantifying A-to-I RNA editing by Sanger sequencing.","authors":"Alla Fishman, Ayelet T Lamm","doi":"10.1016/bs.mie.2024.11.032","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.11.032","url":null,"abstract":"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.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"710 ","pages":"285-302"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143052991","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Assay for ribosome stimulation of angiogenin nuclease activity.

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-04 DOI: 10.1016/bs.mie.2024.11.007

Emily Sholi, Anna B Loveland, Andrei A Korostelev

{"title":"Assay for ribosome stimulation of angiogenin nuclease activity.","authors":"Emily Sholi, Anna B Loveland, Andrei A Korostelev","doi":"10.1016/bs.mie.2024.11.007","DOIUrl":"10.1016/bs.mie.2024.11.007","url":null,"abstract":"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.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"711 ","pages":"381-404"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11839171/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425704","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

cP-RNA-seq for tRNA half sequencing.

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-04 DOI: 10.1016/bs.mie.2024.11.002

Megumi Shigematsu, Justin Gumas, Yohei Kirino

{"title":"cP-RNA-seq for tRNA half sequencing.","authors":"Megumi Shigematsu, Justin Gumas, Yohei Kirino","doi":"10.1016/bs.mie.2024.11.002","DOIUrl":"10.1016/bs.mie.2024.11.002","url":null,"abstract":"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.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"711 ","pages":"135-153"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11938272/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425655","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Analysis of metal-dependent DNA nicking activities by Cas endonucleases.

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-07 DOI: 10.1016/bs.mie.2025.01.034

Giang T Nguyen, Akshara Raju, Dipali G Sashital

引用次数: 0

Bacterial directed evolution of CRISPR base editors.

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-03-08 DOI: 10.1016/bs.mie.2025.01.003

Reilly Q Mach, Shannon M Miller

引用次数: 0

Hydrolytic endonucleolytic ribozyme (HYER): Systematic identification, characterization and potential application in nucleic acid manipulation.

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-03-06 DOI: 10.1016/bs.mie.2025.01.033

Zi-Xian Liu, Jun-Jie Gogo Liu

引用次数: 0

Nanopore sequencing to detect A-to-I editing sites.

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-04 DOI: 10.1016/bs.mie.2024.11.028

Jia Wei Joel Heng, Meng How Tan

引用次数: 0