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The Manipulation of RNA-Guided Nucleic Acid Cleavage with Ninhydrin Chemistry.

ABSTRACT: CRISPR (clustered regularly interspaced short palindromic repeats) systems have been established as valuable genome-editing tools. Controlling CRISPR systems has high biological significance and this field has garnered intense interest. There is a considerable need for simple approaches with no need for protein engineering. The CRISPR systems usually require a guide RNA (gRNA) moiety to recruit and direct the nuclease complexes. In this respect, the ninhydrin (1,2,3-indantrione monohydrate) seems to have considerable potential, as yet unexploited, for modifying gRNA. In this study, ninhydrin chemistry is explored for reversible postsynthetic modification of gRNA molecules. It is further shown that ninhydrin chemistry is efficient in modulating two important CRISPR systems. Thus, ninhydrin chemistry exhibits potential applications in future chemical biology studies.

SUBMITTER: Wang SR

PROVIDER: S-EPMC7341091 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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The Manipulation of RNA-Guided Nucleic Acid Cleavage with Ninhydrin Chemistry.

Wang Shao-Ru SR Huang Hai-Yan HY Liu Jian J Wei Lai L Wu Ling-Yu LY Xiong Wei W Yin Ping P Tian Tian T Zhou Xiang X

Advanced science (Weinheim, Baden-Wurttemberg, Germany) 20200526 13

CRISPR (clustered regularly interspaced short palindromic repeats) systems have been established as valuable genome-editing tools. Controlling CRISPR systems has high biological significance and this field has garnered intense interest. There is a considerable need for simple approaches with no need for protein engineering. The CRISPR systems usually require a guide RNA (gRNA) moiety to recruit and direct the nuclease complexes. In this respect, the ninhydrin (1,2,3-indantrione monohydrate) seem ...[more]

PMID: 32670753

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Project description:2'-Fluoro arabino nucleic acid (FANA), classified as a xeno nucleic acid (XNA), stands as a prominent subject of investigation in synthetic genetic polymers. Demonstrating efficacy as antisense oligonucleotides (ASOs) and exhibiting the ability to fold into functional structures akin to enzymes and aptamers, FANA holds substantial promise across diverse biological and therapeutic domains. Owing to structural similarities to DNA, the utilization of naturally occurring DNA polymerases for DNA-mediated FANA replication is well-documented. In this study, we explore alternative nucleic acid processing enzymes typically employed for DNA oligonucleotide (ON) phosphorylation, ligation, and amplification, and assess their compatibility with FANA substrates. Notably, T4 polynucleotide kinase (T4 PNK) efficiently phosphorylated the 5'-hydroxyl group of FANA using ATP as a phosphate donor. Subsequent ligation of the phosphorylated FANA with an upstream FANA ON was achieved with T4 DNA ligase, facilitated by a DNA splint ON that brings the two FANA ONs into proximity. This methodology enabled the reconstruction of RNA-cleaving FANA 12-7 by ligating two FANA fragments amenable to solid-phase synthesis. Furthermore, Tgo DNA polymerase, devoid of 3' to 5' exonuclease activity [Tgo (exo-)], demonstrated proficiency in performing polymerase chain reaction (PCR) with a mixture of dNTPs and FANA NTPs (fNTPs), yielding DNA-FANA chimeras with efficiency and fidelity comparable to traditional DNA PCR. Notably, T7 RNA polymerase (T7 RNAP) exhibited recognition of double-stranded fA-DNA chimeras containing T7 promoter sequences, enabling in vitro transcription of RNA molecules up to 649 nt in length, even in the presence of highly structured F30 motifs at the 3' end. Our findings significantly expand the enzymatic toolkit for FANA manipulation, encompassing phosphorylation, ligation, chimeric amplification, and templating T7 RNAP-catalyzed RNA transcription. These advancements are poised to expedite fundamental research, functional evolution, and translational applications of FANA-based XNA agents. They also have the potential to inspire explorations of a broader range of non-natural nucleic acids that can be routinely studied in laboratories, ultimately expanding the repertoire of nucleic acid-based biomedicine and biotechnology.

Dataset Information

The Manipulation of RNA-Guided Nucleic Acid Cleavage with Ninhydrin Chemistry.

Publications

The Manipulation of RNA-Guided Nucleic Acid Cleavage with Ninhydrin Chemistry.

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