DNA samples derived from vertebrate skin, bodily cavities and body fluids contain both host microbial DNA; the latter often present as a minor component. Consequently, sequencing of microbiome sample frequently yields reads originating microbe(s) interest, but with vast excess genome-derived reads. In this study, we used methyl-CpG binding domain (MBD) to separate methylated based on differences in CpG methylation density. MBD fused Fc region human antibody (MBD-Fc) binds strongly protein A...

The initiating nucleotide found at the 5' end of primary transcripts has a distinctive triphosphorylated that distinguishes these from all other RNA species. Recognizing this distinction is key to deconvoluting transcriptome plethora processed confound analysis transcriptome. currently available methods do not use targeted enrichment for 5'end transcripts, but rather attempt deplete non-targeted RNA.We developed method, Cappable-seq, directly enriching and enabling determination...

Deaminases have important uses in modification detection and genome editing. However, the range of applications is limited by small number characterized enzymes. To expand toolkit deaminases, we developed an vitro approach that bypasses a major hurdle with their toxicity cells. We assayed 175 putative cytosine deaminases on variety substrates found broad activity double- single-stranded DNA various sequence contexts, including CpG-specific enzymes without preference. also enzyme selectivity...

While all native tRNAs undergo extensive post-transcriptional modifications as a mechanism to regulate gene expression, mapping these remains challenging. The critical barrier is the difficulty of readthrough by reverse transcriptases (RTs). Here we use Induro—a new group-II intron-encoded RT—to map and quantify genome-wide tRNA in Induro-tRNAseq. We show that Induro progressively increases over time selectively overcoming RT stops without altering misincorporation frequency. In parallel...

Dicer ribonucleases of plants and invertebrate animals including Caenorhabditis elegans recognize process a viral RNA trigger into virus-derived small interfering RNAs (siRNAs) to guide specific immunity by Argonaute-dependent interference (RNAi). C. also encodes three Dicer-related helicase (drh) genes closely related the RIG-I-like receptors which initiate broad-spectrum innate against viruses in mammals. Here we developed transgenic strain that expressed intense green fluorescence from...

Abstract With the rapid growth of synthetic messenger RNA (mRNA)-based therapeutics and vaccines, development analytical tools for characterization long, complex RNAs has become essential. Tandem liquid chromatography–mass spectrometry (LC–MS/MS) permits direct assessment mRNA primary sequence modifications thereof without conversion to cDNA or amplification. It relies upon digestion with site-specific endoribonucleases generate pools short oligonucleotides that are then amenable MS-based...

We have isolated three naturally occurring strains of Turnip crinkle virus (TCV) that break resistance in Di-17 Arabidopsis. Two mutations the N terminus TCV coat protein, D4N and P5S, were shown to confer this phenotype. Thus, region protein is involved eliciting responses

Since its initial characterization, Escherichia coli RNase I has been described as a single-strand specific RNA endonuclease that cleaves substrate in largely sequence independent manner. Here, we describe strong calcium (Ca2+)-dependent activity of on double-stranded (dsRNA), and Ca2+-dependent novel hybridase activity, digesting the strand DNA:RNA hybrid. Surprisingly, Ca2+ does not affect single stranded (ssRNA), suggesting role for modulation activity. Mutation previously overlooked...

Genome-wide investigations have dramatically increased our understanding of nucleosome positioning and the role chromatin in gene regulation, yet some genomic regions been poorly represented human maps. One such region is by chromosome 9p21-22, which contains type I interferon cluster that includes 16 alpha genes single beta, epsilon, omega genes. A high-density mapping strategy was used to generate locus-wide maps organization this biomedically important locus at a steady state during time...

We report a target enrichment method to map nucleosomes of large genomes at unprecedented coverage and resolution by deeply sequencing locus-specific mononucleosomal DNA enriched via hybridization with bacterial artificial chromosomes. achieved ∼10 000-fold specific loci, which enabled up ∼500-fold higher than has been reported in mammalian genome. demonstrate the advantages generating high-sequencing for mapping center discrete nucleosomes, we show use during T cell differentiation using...

Nucleosome positioning on the chromatin strand plays a critical role in regulating accessibility of DNA to transcription factors and modifying enzymes. Hence, detailed information nucleosome depletion or movement at cis -acting regulatory elements has potential identify predicted binding sites for trans factors. Using novel method based enrichment mononucleosomal by bacterial artificial chromosome hybridization, we mapped positions deep sequencing across 250 kb, encompassing cystic fibrosis...

Variation in the distribution of methylated CpG (methyl-CpG) genomic DNA (gDNA) across tree life is biologically interesting and useful studies. We illustrate use human methyl-CpG-binding domain (MBD2) to fractionate angiosperm into eukaryotic nuclear (methyl-CpG-rich) vs. organellar prokaryotic (methyl-CpG-poor) elements for metagenomic sequencing projects. •MBD2 has been used enrich animal systems. Using gDNA from five model species, we apply a similar approach identify whether MBD2 can...

Abstract Cytosine deaminases have important uses in the detection of epigenetic modifications and genome editing. However, range applications is limited by a small number well characterized enzymes. To expand toolkit deaminases, we developed an in-vitro approach that bypasses major hurdle with their severe toxicity expression hosts. We systematically assayed activity 175 putative cytosine on unprecedented variety substrates epigenetically relevant base modifications. found enzymes high...

Abstract “Microbiome” is used to describe the communities of microorganisms and their genes in a particular environment, including association with eukaryotic host or part host. One challenge microbiome analysis concerns presence DNA samples. Removal before sequencing results greater sequence depth intended target population. This unit describes novel method microbial enrichment which methylated such as human genomic selectively bound separated from next‐generation (NGS) library...

Abstract We have developed Cappable-seq that specifically captures primary RNA transcripts by enzymatically modifying the 5’ triphosphorylated end of with a selectable tag. first applied to E. coli , achieving up 50 fold enrichment and identifying an unprecedented 16539 transcription start sites (TSS) genome-wide at single base resolution. also mouse cecum sample for time identified TSS in microbiome. Furthermore, universally depletes ribosomal reduces complexity transcriptome quantifiable...

The chemical modification of RNA bases represents a ubiquitous activity that spans all domains life. Pseudouridylation is the most common and observed within tRNA, rRNA, ncRNA mRNAs. Pseudouridine synthase or ‘PUS’ enzymes include those rely on guide molecules others function as ‘stand-alone’ enzymes. Among latter, several have been shown to modify mRNA transcripts. Although recent studies defined structural requirements for act PUS target, mechanisms by which PUS1 recognizes these target...

Combinations of ribonucleases (RNases) are commonly used to digest RNA into oligoribonucleotide fragments prior liquid chromatography-mass spectrometry (LC-MS) analysis. The distribution the RNase target sequences or nucleobase sites within an molecule is critical for achieving a high mapping coverage. Cusativin and MC1 nucleotide-specific endoribonucleases encoded in cucumber bitter melon genomes, respectively. Their specificity cytidine (Cusativin) uridine (MC1) make them ideal molecular...

High temperature stress is a major environmental factor for all photosynthetic organisms.Some lichen species could have the ability of tolerance against global warming.In present study, we investigated effects high on GABA metabolism in two different Evernia prunastri and Usnea sp.. sp. were collected from unpolluted locations Bilecik, TURKEY.Evernia kept at 45 °C 0, 24 48 h.We analyzed content, glutamate dehydrogenase (GDH) decarboxylase (GAD) activities also chlorophyll MDA contents thalli...

Monitoring the dynamic changes of cellular tRNA pools is challenging, due to extensive post-transcriptional modifications individual species. The most critical component in tRNAseq a processive reverse transcriptase (RT) that can read through each modification with high efficiency. Here we show recently developed group-II intron RT Induro has processivity and efficiency necessary profile dynamics. Using our Induro-tRNAseq, simpler more comprehensive than best methods date, progressively...

Next Generation Sequencing (NGS) has significantly impacted human genetics, enabling a comprehensive characterization of the genome as well better understanding many genomic abnormalities. By delivering massive DNA sequences at unprecedented speed and cost, NGS promises to make personalized medicine reality in foreseeable future. To date, library construction with clinical samples been challenge, primarily due limited quantities sample available. Our objective here was overcome this...

The SARS-CoV-2 viral genome contains a positive-strand single-stranded RNA of ∼30 kb. Human ACE2 protein is the receptor for virus attachment and infection. We propose to use ribonucleases (RNases) as antiviral agents destroy in vitro. In virions, protected by capsid proteins, membrane nucleocapsid proteins. To utilize RNases strategy, we set out construct RNase fusion with human N-terminal domain (ACE2NTD). expressed six proteins E. coli cells: (1) MBP-ACE2NTD, (2) ACE2NTD-GFP, (3) I...