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Steric conflicts of the 2 -OH groups in silico introduced into the AvaII and EcoO109I (13) dsDNA complexes. 2 -OH groups were grafted onto the 2 -deoxyriboses, without alteration of their puckers, with the help of the CNS program (30). Maximal and cumulative clashes were then scored as in a recent analysis of methyl group steric conflicts (32). The 1.1 ˚ A threshold for functionally relevant steric clashes refers to methyl groups on DNA bases, but we suspect that a similar threshold applies to the clashes of 2 -OH groups (red horizontal line). For each nucleotide maximal and cumulative clash is indicated. Additionally the clashes with nucleic acid and protein were independently summed. Values were averaged for both DNA strands. The AvaII and EcoO109I target sequences are shaded in sepia.
Source publication
Restriction endonucleases naturally target DNA duplexes. Systematic screening has identified a small minority of these enzymes that can also cleave RNA/DNA heteroduplexes and that may therefore be useful as tools for RNA biochemistry. We have chosen AvaII (G↓GWCC, where W stands for A or T) as a representative of this group of restriction endonucle...
Contexts in source publication
Context 1
... specific complex was not formed for any of the mutants and non-cognate sequences except for trace amounts observed for the variants containing the E115Q substitution (Supplementary Figure S8). Comparisons of the wild-type AvaII and its variants in the presence of EDTA were not informative, because the specific complex did not form even with the wild-type enzyme (Supplementary Figure S9). This is not surprising, since the EDTA chelates out not only the metal ion close to the central base pair, but also the active site metals, which are known to make a major contribution to DNA affinity of PD-(D/E)XK restriction endonucleases. ...
Citations
... H. pylori has been derived from a variety of clinical specimens and identified using a multiplex polymerase chain reaction (Multiplex-PCR). Several virulence genes in H. pylori isolates have been reported, including Vacuolating cytotoxin A (VacA) 17 , cytotoxin-associated A (cagA) 17 , restriction endonuclease A (IceA) 18 , Outer inflammatory protein A (OipA) 19 , and blood-group antigen-binding adhesin (BabA2) 19 . These pathogenicity genes may have a role in the progression of H. pylori. ...
Even though Helicobacter pylori (H. pylori) is a serious pathogen, its origin is unknown. Poultry (Chicken, Turkey, Quebec, Goose, and Ostrich) are consumed as a regular protein source by a large number of people across the world; therefore, sanitary ways of delivering poultry for food are important for global health. As a result, we looked at the distribution of the pathogenicity cagA, vacA, babA2, oipA, and iceA in H. pylori isolates in poultry meat, as well as their antimicrobial resistance. Wilkins Chalgren anaerobic bacterial medium was used to cultivate 320 raw poultry specimens. Disk diffusion and Multiplex-PCR were used to investigate antimicrobial resistance and genotyping patterns, separately. H. pylori was found in 20 of 320 (6.25%) raw poultry samples. The highest incidence of H. pylori was found in chicken raw meat (15%), whereas the fewest was found in Goose and Quebec (0.00%). Resistance to ampicillin (85%), tetracycline (85%), and amoxicillin (75%) were greatest in H. pylori isolates. The percentage of H. pylori isolates with a MAR value of more than 0.2 was 17/20 (85%). The most prevalent genotypes discovered were VacA s1a (75%), m1a (75%), s2 (70%) and m2 (65%), and cagA (60%). The most typically discovered genotype patterns were s1am1a (45%), s2m1a (45%), and s2m2 (30%). BabA2, OipA + , and OipA− genotypes were found in 40%, 30%, and 30% of the population. In summary, the poultry flesh was polluted by H. pylori, with the babA2, vacA, and cagA genotypes being more prevalent. The simultaneous occurrence of vacA, cagA, iceA, oipA, and babA2 genotypes in antibiotic-resistant H. pylori bacteria implies a serious public health concern about raw poultry eating. In the future, researchers should look into H. pylori's resistance to multiple antibacterial drugs in Iran.
... Endonuclease function in many organisms is involved in DNA damage repair mechanisms, restricting gene expression and antiviral responses to infection in bacteria 1 . Endonucleases that target RNA substrates are involved in mRNA splicing, RNA silencing pathways, maturation of t-RNAs, and other non-coding RNAs [2][3][4][5] . Adapting and engineering these natural enzyme catalysts has led to cornerstone biotechnologies from recombinant DNAs to gene editing technologies [6][7][8] . ...
... Our work lays the groundwork for new versions with enhanced targeting. 4 . Created with BioRender.com. ...
Endonucleases are enzymes that cleave internal phosphodiester bonds within double-stranded DNA or RNA and are essential for biological functions. Herein, we use genetic code expansion to create an unnatural endonuclease that cleaves non-coding RNAs including short interfering RNA (siRNA) and microRNAs (miRNAs), a function that does not exist in nature. We introduce a metal-chelating unnatural amino acid, (2,2′-bipyridin-5-yl)alanine (BpyAla) to impart endonuclease activity to the viral suppressor of RNA silencing protein p19. Upon binding of copper, the mutant p19-T111BpyAla displays catalytic site-specific cleavage of siRNA and human miRNAs. Catalysis is confirmed using fluorescence polarization and fluorescence turn-on. Global miRNA profiling reveals that the engineered enzyme cleaves miRNAs in a human cell line. The therapeutic potential is demonstrated by targeting miR-122, a critical host factor for the hepatitis C virus (HCV). Unnatural endonuclease function is shown to deplete miR-122 levels with similar effects to an antagomir that reduces HCV levels therapeutically.
... H. pylori pathogenesis is linked to virulence genes only found in certain groups. Several virulence genes in H. pylori isolates have been reported, including vacuolating cytotoxin A (VacA) [23], cytotoxin-associated A (CagA) [23], restriction endonuclease A (IceA) [24], outer inflammatory protein A (OipA) [25], and bloodgroup antigen-binding adhesin (BabA2) [25]. Among the most efficient ways for studying relationships between H. pylori strains from diverse samples is genotyping using the virulence genes (cagA, vacA, babA2, oipA, and iceA) [15]. ...
Even though Helicobacter pylori (H. pylori) is a serious pathogen, its origin is unknown. Poultry (chicken, turkey, quail, goose, and ostrich) is consumed as a regular protein source by many people across the world; therefore, sanitary ways of delivering poultry for food are important for global health. As a result, the distribution of the virulence genes cagA, vacA, babA2, oipA, and iceA in H. pylori isolates in poultry meat, as well as their antibacterial resistance, was investigated. A Wilkins Chalgren anaerobic bacterial medium was used to cultivate 320 samples of raw poultry meat. Disk diffusion and multiplex-PCR were used to investigate both antimicrobial resistance and genotyping patterns. H. pylori was found in 20 of 320 (6.25 %) raw chicken meat samples. The highest incidence of H. pylori was found in chicken raw meat (15%), whereas no isolate was recovered from goose or quail raw meat (0.00%). Resistance to ampicillin (85%), tetracycline (85%), and amoxicillin (75%) were the most commonly detected in the tested H. pylori isolates. The percentage of H. pylori isolates with a multiple antibiotic resistance (MAR) index value of more than 0.2 was 17/20 (85%). The most prevalent genotypes detected were VacA (75%), m1a (75%), s2 (70%) and m2 (65%), and cagA (60%). The most typically detected genotype patterns were s1am1a (45 %), s2m1a (45 %), and s2 m2 (30 %). babA2, oipA+, and oipA- genotypes were found in 40%, 30%, and 30% of the population, respectively. In summary, fresh poultry meat was polluted by H. pylori, with the babA2, vacA, and cagA genotypes being more prevalent. The simultaneous occurrence of vacA, cagA, iceA, oipA, and babA2 genotypes in antibiotic-resistant H. pylori bacteria raises a serious public health concern regarding the consumption of raw poultry. Future research should evaluate antimicrobial resistance among H. pylori isolates in Iran.
... This needs to be avoided by replacing the type II restriction enzyme with type IIS restriction enzymes. 46,126 mRNA purification. IVT mRNAs are mixed with RNA polymerase and DNA templates after synthesis; thus, it is essential to purify IVT mRNA, including removing immunostimulatory contaminants, free ribonucleotides, short mRNA and DNA templates. ...
The therapeutic use of messenger RNA (mRNA) has fueled great hope to combat a wide range of incurable diseases. Recent rapid advances in biotechnology and molecular medicine have enabled the production of almost any functional protein/peptide in the human body by introducing mRNA as a vaccine or therapeutic agent. This represents a rising precision medicine field with great promise for preventing and treating many intractable or genetic diseases. In addition, in vitro transcribed mRNA has achieved programmed production, which is more effective, faster in design and production, as well as more flexible and cost-effective than conventional approaches that may offer. Based on these extraordinary advantages, mRNA vaccines have the characteristics of the swiftest response to large-scale outbreaks of infectious diseases, such as the currently devastating pandemic COVID-19. It has always been the scientists’ desire to improve the stability, immunogenicity, translation efficiency, and delivery system to achieve efficient and safe delivery of mRNA. Excitingly, these scientific dreams have gradually been realized with the rapid, amazing achievements of molecular biology, RNA technology, vaccinology, and nanotechnology. In this review, we comprehensively describe mRNA-based therapeutics, including their principles, manufacture, application, effects, and shortcomings. We also highlight the importance of mRNA optimization and delivery systems in successful mRNA therapeutics and discuss the key challenges and opportunities in developing these tools into powerful and versatile tools to combat many genetic, infectious, cancer, and other refractory diseases.
... 14 In the presence of magnesium, their intrinsic enzymatic activities cleave the phosphodiester backbone of targeted mRNAs. 15 However, since the trans-cleaving HHRz is easily degraded because of its structural instability, the scaffolds can be introduced to deliver structural stability and maintain stability in vivo. 16 In the nucleus, small nucleolar RNAs (snoRNAs) are 60-300-nucleotide-long noncoding RNAs that accumulate in the nucleolus, regulating ribose 2′-OH hydroxyl methylation (2′-O-Me) and conversion of uridine to pseudouridine modifications of RNAs, transfer RNAs (tRNAs), and snRNAs. ...
Trans‐cleaving techniques have been most enthusiastically embraced in the development of therapy for genetic diseases, particularly in the correction of monogenic recessive mutations at the messenger RNA level. However, easy degradation and poor catalytic activity in vivo remain significant obstacles to trans‐cleaving of the hammerhead ribozyme. Herein, we found a novel scaffold RNA that stabilizes the ribozyme structure in trans‐cleaving and promotes the knockdown efficiency of the hammerhead ribozyme in specific regions of living cells. We can give the trans‐cleaving hammerhead ribozyme the ability to knock down specific genes in specific cell regions by changing different scaffolds. Therefore, our study proves the potential usefulness of the RNA knockdown strategy with high‐specific trans‐cleaving hammerhead ribozyme as a therapeutic approach in gene therapy.
