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National Science Review Aug 2023Current DNA base editors contain nuclease and DNA deaminase that enables deamination of cytosine (C) or adenine (A), but no method for guanine (G) or thymine (T) editing...
Current DNA base editors contain nuclease and DNA deaminase that enables deamination of cytosine (C) or adenine (A), but no method for guanine (G) or thymine (T) editing is available at present. Here we developed a deaminase-free glycosylase-based guanine base editor (gGBE) with G editing ability, by fusing Cas9 nickase with engineered N-methylpurine DNA glycosylase protein (MPG). By several rounds of MPG mutagenesis via unbiased and rational screening using an intron-split EGFP reporter, we demonstrated that gGBE with engineered MPG could increase G editing efficiency by more than 1500 fold. Furthermore, this gGBE exhibited high base editing efficiency (up to 81.2%) and high G-to-T or G-to-C (i.e. G-to-Y) conversion ratio (up to 0.95) in both cultured human cells and mouse embryos. Thus, we have provided a proof-of-concept of a new base editing approach by endowing the engineered DNA glycosylase the capability to selectively excise a new type of substrate.
PubMed: 37404457
DOI: 10.1093/nsr/nwad143 -
Nucleic Acids Research Oct 2023Xeno-nucleic acids (XNAs) have gained significant interest as synthetic genetic polymers for practical applications in biomedicine, but very little is known about their...
Xeno-nucleic acids (XNAs) have gained significant interest as synthetic genetic polymers for practical applications in biomedicine, but very little is known about their biophysical properties. Here, we compare the stability and mechanism of acid-mediated degradation of α-l-threose nucleic acid (TNA) to that of natural DNA and RNA. Under acidic conditions and elevated temperature (pH 3.3 at 90°C), TNA was found to be significantly more resistant to acid-mediated degradation than DNA and RNA. Mechanistic insights gained by reverse-phase HPLC and mass spectrometry indicate that the resilience of TNA toward low pH environments is due to a slower rate of depurination caused by induction of the 2'-phosphodiester linkage. Similar results observed for 2',5'-linked DNA and 2'-O-methoxy-RNA implicate the position of the phosphodiester group as a key factor in destabilizing the formation of the oxocarbenium intermediate responsible for depurination and strand cleavage of TNA. Biochemical analysis indicates that strand cleavage occurs by β-elimination of the 2'-phosphodiester linkage to produce an upstream cleavage product with a 2'-threose sugar and a downstream cleavage product with a 3' terminal phosphate. This work highlights the unique physicochemical properties available to evolvable non-natural genetic polymers currently in development for biomedical applications.
PubMed: 37650628
DOI: 10.1093/nar/gkad716 -
Nucleosides, Nucleotides & Nucleic Acids May 2024The -methyladenosine (mA) epigenetic modification exists in many RNAs and is related to many human diseases. Chemically synthesized RNAs containing the modification are...
The -methyladenosine (mA) epigenetic modification exists in many RNAs and is related to many human diseases. Chemically synthesized RNAs containing the modification are required for projects aimed at studying biological processes, mechanisms, and pathogenesis related to mA. Existing methods for the synthesis of mA containing RNAs use tetrabutylammonium fluoride (TBAF) for the deprotection of the 2'-silyl protecting groups. Since TBAF is nonvolatile, and is relatively non-polar, its use in the desilylation of RNA requires repeated desalting, which is tedious and gives low yields. Here we report the use of the volatile and neat triethylamine hydrogen fluoride (TEA-HF) for desilylation of mA RNA synthesis. We found that the method is much simpler, and-in our hands-give significantly higher yield of RNA. Two major concerns for mA RNA synthesis are depurination and Dimroth rearrangement. HPLC and MALDI MS of the RNA indicated that depurination is not a problem for the new method. The absence of Dimroth rearrangement is proven by RNA digestion followed by HPLC analysis of the nucleosides.
PubMed: 38735066
DOI: 10.1080/15257770.2024.2353181 -
Molecules (Basel, Switzerland) Mar 2024The plant-derived toxin ricin is classified as a type 2 ribosome-inactivating protein (RIP) and currently lacks effective clinical antidotes. The toxicity of ricin is...
The plant-derived toxin ricin is classified as a type 2 ribosome-inactivating protein (RIP) and currently lacks effective clinical antidotes. The toxicity of ricin is mainly due to its ricin toxin A chain (RTA), which has become an important target for drug development. Previous studies have identified two essential binding pockets in the active site of RTA, but most existing inhibitors only target one of these pockets. In this study, we used computer-aided virtual screening to identify a compound called RSMI-29, which potentially interacts with both active pockets of RTA. We found that RSMI-29 can directly bind to RTA and effectively attenuate protein synthesis inhibition and rRNA depurination induced by RTA or ricin, thereby inhibiting their cytotoxic effects on cells in vitro. Moreover, RSMI-29 significantly reduced ricin-mediated damage to the liver, spleen, intestine, and lungs in mice, demonstrating its detoxification effect against ricin in vivo. RSMI-29 also exhibited excellent drug-like properties, featuring a typical structural moiety of known sulfonamides and barbiturates. These findings suggest that RSMI-29 is a novel small-molecule inhibitor that specifically targets ricin toxin A chain, providing a potential therapeutic option for ricin intoxication.
Topics: Animals; Mice; Ricin; Ribosome Inactivating Proteins, Type 2; Drug Development; Hydrolases; Liver
PubMed: 38611715
DOI: 10.3390/molecules29071435 -
Nucleosides, Nucleotides & Nucleic Acids 2024This work catalogued oligonucleotide sequences and sequence compositions based on the overall yield of full-length product obtained by the phosphoramidite...
This work catalogued oligonucleotide sequences and sequence compositions based on the overall yield of full-length product obtained by the phosphoramidite chemistry-based solid phase synthesis. In total, 76 sequences with different dinucleotide and trinucleotide repeats were synthesized, and the fully-deprotected products were analyzed by denaturing anion exchange HPLC. Overall, sequences containing more 2'-deoxyadenosine residues were obtained in relatively lower yields, likely due to the relative ease of 2'-deoxyadenosine to undergo depurination during the detritylation reaction. Furthermore, dinucleotide steps, such as d(CG)/d(GC) and d(AG)/d(GA), likely contribute the overall lower yields of full-length products as well.
Topics: Solid-Phase Synthesis Techniques; Organophosphorus Compounds; Deoxyribonucleotides; Base Sequence; Oligonucleotides; Chromatography, High Pressure Liquid
PubMed: 38116988
DOI: 10.1080/15257770.2023.2295478 -
RNA Biology Jan 2024RNA modifications, including -7-methylguanosine (mG), are pivotal in governing RNA stability and gene expression regulation. The accurate detection of internal mG...
RNA modifications, including -7-methylguanosine (mG), are pivotal in governing RNA stability and gene expression regulation. The accurate detection of internal mG modifications is of paramount significance, given recent associations between altered mG deposition and elevated expression of the methyltransferase METTL1 in various human cancers. The development of robust mG detection techniques has posed a significant challenge in the field of epitranscriptomics. In this study, we introduce two methodologies for the global and accurate identification of mG modifications in human RNA. We introduce borohydride reduction sequencing (Bo-Seq), which provides base resolution mapping of mG modifications. Bo-Seq achieves exceptional performance through the optimization of RNA depurination and scission, involving the strategic use of high concentrations of NaBH, neutral pH and the addition of 7-methylguanosine monophosphate (mGMP) during the reducing reaction. Notably, compared to NaBH-based methods, Bo-Seq enhances the mG detection performance, and simplifies the detection process, eliminating the necessity for intricate chemical steps and reducing the protocol duration. In addition, we present an antibody-based approach, which enables the assessment of mG relative levels across RNA molecules and biological samples, however it should be used with caution due to limitations associated with variations in antibody quality between batches. In summary, our novel approaches address the pressing need for reliable and accessible methods to detect RNA mG methylation in human cells. These advancements hold the potential to catalyse future investigations in the critical field of epitranscriptomics, shedding light on the complex regulatory roles of mG in gene expression and its implications in cancer biology.
Topics: Humans; RNA; Nucleotides; Methylation; Methyltransferases; RNA Processing, Post-Transcriptional; Guanosine
PubMed: 38566310
DOI: 10.1080/15476286.2024.2337493 -
The Analyst Jun 2024The study aimed to analyze nusinersen metabolites in the cerebrospinal fluid samples using ion-pair reversed-phase ultrahigh-performance liquid chromatography coupled...
Study of nusinersen metabolites in the cerebrospinal fluid of children with spinal muscular atrophy using ultra-high-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry.
The study aimed to analyze nusinersen metabolites in the cerebrospinal fluid samples using ion-pair reversed-phase ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. Three different sample preparation methods were tested for extraction and purification, but solid phase extraction appeared to be the most suitable, allowing a significant sample enrichment (40-fold). This step was necessary to detect and identify metabolites of nusinersen in the cerebrospinal fluid. The developed and applied analytical procedure enabled the identification of nusinersen metabolites: sequences shorter by several nucleotides from the 3' end; shorter by several nucleotides from both the 3' and 5' ends; and some depurination products. To the best of our knowledge, this is the first report on the analysis and identification of nusinersen metabolites in cerebrospinal fluid samples taken from children with spinal muscular atrophy treated with Spinraza.
PubMed: 38828890
DOI: 10.1039/d4an00436a -
Toxicology Letters May 2024Ricin toxin (RT) is highly cytotoxic and can release a considerable amount of pro-inflammatory factors due to depurination, causing excessive inflammation that may...
Ricin toxin (RT) is highly cytotoxic and can release a considerable amount of pro-inflammatory factors due to depurination, causing excessive inflammation that may aggravate the harm to the body. Pyroptosis, a type of gasdermin-mediated cell death, is a contributor to the exacerbation of inflammation. Accumulating evidence indicate that pyroptosis plays a significant role in the pathogen infection and tissue injury, suggesting a potential correlation between pyroptosis and RT-induced inflammation. Here, we aim to demonstrate this correlation and explore its molecular mechanisms. Results showed that RT triggers mouse alveolar macrophage MH-S cells pyroptosis by activating caspase-3 and cleaving Gasgermin E (GSDME). In contrast, inhibition of caspase-3 with Z-DEVD-FMK (inhibitor of caspase-3) or knockdown of GSDME attenuates this process, suggesting the essential role of caspase-3/GSDME-mediated pyroptosis in contributing to RT-induced inflammation. Collectively, our study enhances our understanding of a novel mechanism of ricin cytotoxicity, which may emerge as a potential target in immunotherapy to control the RT-induced inflammation.
Topics: Pyroptosis; Ricin; Animals; Mice; Caspase 3; Inflammation; Cell Line; Macrophages, Alveolar; Gasdermins
PubMed: 38642674
DOI: 10.1016/j.toxlet.2024.04.007 -
Gene Aug 2023Ribosome-inactivating proteins (RIPs) are RNA N-glycosidases that depurinate an adenine residue in the conserved alpha-sarcin/ricin loop (SRL) of rRNA, inhibiting...
Ribosome-inactivating proteins (RIPs) are RNA N-glycosidases that depurinate an adenine residue in the conserved alpha-sarcin/ricin loop (SRL) of rRNA, inhibiting protein synthesis. Previously, we reported the existence of these toxins in insects, whose presence is restricted to mosquitoes from the Culicinae subfamily (e.g., Aedes aegypti) and whiteflies from the Aleyrodidae family (e.g., Bemisia tabaci). Both groups of genes are derived from two independent horizontal gene transfer (HGT) events and are evolving under purifying selection. Here, we report and characterize the occurrence of a third HGT event in the Sciaroidea superfamily, which supports the recurrent acquisition of RIP genes by insects. Transcriptomic experiments, available in databases, allowed us to describe the temporal and spatial expression profiles for these foreign genes in these organisms. Furthermore, we found that RIP expression is induced after infection with pathogens and provided, for the first time, transcriptomic evidence of parasite SRL depurination. This evidence suggests a possible role of these foreign genes as immune effectors in insects.
Topics: Animals; Ribosome Inactivating Proteins; Gene Transfer, Horizontal; Insecta; Protein Biosynthesis; RNA, Ribosomal; Ricin; Hemiptera; Plant Proteins
PubMed: 37286020
DOI: 10.1016/j.gene.2023.147547 -
Biochimie Jul 2024Understanding the diversity of DNA structure and functions in biology requires tools to study this biomolecule selectively and thoroughly. Fluorescence methods are...
Understanding the diversity of DNA structure and functions in biology requires tools to study this biomolecule selectively and thoroughly. Fluorescence methods are powerful technique for non-invasive research. Due to the low quantum yield, the intrinsic fluorescence of nucleotides has not been considered for use in the detection and differentiation of nucleic acid bases. Here, we have studied the influence of protonation of nucleotides on their fluorescence properties. We show that protonation of ATP and GTP leads to enhanced intrinsic fluorescence. Fluorescence enhancement at acidic pH has been observed for double-stranded DNA and single-stranded oligonucleotides. The formation of G4 secondary structures apparently protected certain nucleotides from protonation, resulting in less pronounced fluorescence enhancement. Furthermore, acid-induced depurination under protonation was less noticeable in G4 structures than in double-stranded and single-stranded DNA. We show that changes in the intrinsic fluorescence of guanine can be used as a sensitive sensor for changes in the structure of the DNA and for the protonation of specific nucleotides.
Topics: Guanine; Protons; DNA; Guanosine Triphosphate; Hydrogen-Ion Concentration; Fluorescence; Spectrometry, Fluorescence; DNA, Single-Stranded; Adenosine Triphosphate; Nucleic Acid Conformation; G-Quadruplexes
PubMed: 38447859
DOI: 10.1016/j.biochi.2024.03.003