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Nucleic Acids as Therapeutic Targets and Agents
Published in David E. Thurston, Ilona Pysz, Chemistry and Pharmacology of Anticancer Drugs, 2021
Peter Dervan, working at the California Institute of Technology (Pasadena, USA), was one of the first to recognize that it may be possible to design small-molecule minor-groove binding agents that may be able to “read” DNA sequence. His ideas were based on the natural products netropsin and distamycin (Figure 5.62) produced by Streptomyces netropsis and distallicus, respectively. William Lown from the University of Alberta (Canada) developed a family of similar molecules known as the Lexitropsins. Structures of the natural products netropsin and distamycin that bind in the minor groove of DNA recognizing AT bases.
Ultraviolet and Light Absorption Spectrometry
Published in Adorjan Aszalos, Modern Analysis of Antibiotics, 2020
Zoltan M. Dinya, Ferenc J. Sztaricskai
Of the antibiotics with a pyrrole skeleton, netropsin, coumermycins, prodigiosins, verrucarin E, anisomycin, and pyrrolnitrin are the most important. Among these compounds only pyrrolnitrin (107) achieved actual pharmaceutical use. Pyrrolnitrin exhibits absorption at 252 nm (ε max 750 m2/mol) in ethanol solution, and at the same time simple pyrrole derivatives usually display a more intense band in the range 240—270 nm. The antibiotics containing more than one pyrrole unit (such as prodigiosin) show intense absorption in the visible range: at 460—470 nm in alkaline solution and between 520 and 540 nm in acid medium.
Potential of CRISPR/Cas system in the diagnosis of COVID-19 infection
Published in Expert Review of Molecular Diagnostics, 2021
V. Edwin Hillary, Savarimuthu Ignacimuthu, S. Antony Ceasar
CRISPR/Cas9 system has been adopted by the scientific community for the diagnosis of infectious diseases (Table 2). Pardee et al. [42] first demonstrated a simple novel method by combining CRISPR/Cas9 with isothermal amplification technique referred to as nucleic acid sequence-based amplification (NASBA) and detected Zika virus strain. The researchers utilized the dsDNA to intermediate the NASBA that served as a substrate for the Cas9 endonuclease. The sgRNA-Cas9 complex cleaves the resulting dsDNA, full-length strand (not truncated strands) activated the toehold switch sensors and led to color changes in order to identify different strains. From the result, investigators stated that this biomolecular platform resolved practical limitations and suggested that this could be used to develop molecular diagnostics for challenging the pandemic [42]. Guk et al. [41] developed a CRISPR/Cas9-mediated fluorescent in situ hybridization (DNA-FISH) method to detect methicillin-resistant Staphylococcus aureus (MRSA) [41]. In this method, the dCas9/sgRNA complex coupled with an SYBR green I fluorescent probe was used to target the mecA gene and recognize mecA gene associated with MRSA. The dCas9/sgRNA did not induce DNA cleavage but it recognizes the target DNA sequence, thus it is suitable for detection by FISH. The corresponding fluorescence was sufficient to efficiently detect the MRSA even at a lower concentration of 10 CFU/ml. From the result, researchers reported that dCas9/sgRNA-based detection is simple to detect various pathogens [41]. In another study, a combination of CRISPR/Cas9 with optical DNA mapping was employed to diagnose bacterial antibiotic-resistance genes [60]. In this demonstration, Cas9 and gRNA were fused that cleaved the targeted specific DNA sequences of resistance genes. Then fluorescent dye (YOYO-1) and netropsin bound to the resulting DNA based on AT-rich regions and detected each DNA segment of different resistant genes. The addition of multiple crRNA detected many different genes in each reaction. From these results, Muller et al. [60] concluded that CRISPR/Cas9 system in combination with optical DNA mapping could be applied for the detection of different pathogens (Figure 1).
CRISPR-cas systems based molecular diagnostic tool for infectious diseases and emerging 2019 novel coronavirus (COVID-19) pneumonia
Published in Journal of Drug Targeting, 2020
Xiaohong Xiang, Keli Qian, Zhen Zhang, Fengyun Lin, Yang Xie, Yang Liu, Zongfa Yang
Type II CRISPR-Cas9 based technology has been reported by several groups for infectious disease diagnostics [23,24,39]. Pardee et al. developed a novel method that combine CRISPR-Cas9 with an isothermal amplification technique called NASBA (nucleic acid sequence-based amplification) to differentiate ZIKV strains in single‐base discrimination [23]. The investigators exploited the (ds)DNA, an intermediate of the NASBA amplification process, which serve as substrate for the Cas9 endonuclease. sgRNA-Cas9 complex cleave the resulting dsDNA, resulting in the truncated or full-length DNA fragments formed upon Cas9 cleavage with or without a strain-specific PAM, respectively. Full-length strands but not the truncated DNA fragments triggered the toehold switch, leading to a colour change to distinguish the different strains. Guk et al. developed a method to detect methicillin‐resistant Staphylococcus aureus (MRSA) via combination CRISPR-Cas9 with FISH（DNA fluorescent in situ hybridization) [24]. In this method, dCas9/sgRNA complex targets and recognises target mecA gene, which is associated with methicillin resistance in MRSA [40]. dCas9 does not induce DNA cleavage when the dCas9/sgRNA complex recognises the target DNA sequence, which can be detected by FISH and the corresponding fluorescence intensity reflects the concentration of MRSA. It’s easy to detect MRSA at a detection concentration of 10 CFU/ml and rapid distinguish between S. aureus isolates in the presence or absence of mecA gene. However, a new mecA homologue mecALGA251 shared 70% nucleotide homology with mecA [41], which results in possible false negative results by using this method. Meanwhile, the detection of the mecA gene is not specific to MRSA, because a small proportion of methicillin‐susceptible Staphylococcus aureus (MSSA) with mecA gene and a large proportion of MRSA strains lack of mecA [42,43]. Combination CRISPR-Cas9 with optical DNA mapping was also applied to identify bacterial antibiotic resistance genes [39]. In this assay, Cas9/gRNA complex specifically recognises and cleaves nucleic acid sequences of resistance genes. Then fluorescent dye (YOYO-1) and netropsin independently and selectively binds to the resulting DNA based on AT-rich regions, resulting in a difference emission intensity allows to detect different resistance genes.