Fluorescent analogs of cyclic and linear dinucleotides as phosphodiesterase and oligoribonuclease activity probes

Zhou, Jie; Zheng, Yue; Roembke, Benjamin T.; Robinson, Sarah; Opoku‐Temeng, Clement; Sayre, D.; Sintim, Herman O.

doi:10.1039/c6ra25394f

Cited by 11 publications

(1 citation statement)

References 58 publications

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“…To determine the polarity of NrnA Bs we used a fluorescence-based assay as described previously ( 37 ). This assay is based on the differential fluorescence output of the nucleotide analog 2-aminopurine.…”

Section: Methodsmentioning

confidence: 99%

NrnA is a 5′-3′ exonuclease that processes short RNA substrates in vivo and in vitro

Weiss

Myers

et al. 2022

Nucleic Acids Research

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Bacterial RNases process RNAs until only short oligomers (2–5 nucleotides) remain, which are then processed by one or more specialized enzymes until only nucleoside monophosphates remain. Oligoribonuclease (Orn) is an essential enzyme that acts in this capacity. However, many bacteria do not encode for Orn and instead encode for NanoRNase A (NrnA). Yet, the catalytic mechanism, cellular roles and physiologically relevant substrates have not been fully resolved for NrnA proteins. We herein utilized a common set of reaction assays to directly compare substrate preferences exhibited by NrnA-like proteins from Bacillus subtilis, Enterococcus faecalis, Streptococcus pyogenes and Mycobacterium tuberculosis. While the M. tuberculosis protein specifically cleaved cyclic di-adenosine monophosphate, the B. subtilis, E. faecalis and S. pyogenes NrnA-like proteins uniformly exhibited striking preference for short RNAs between 2–4 nucleotides in length, all of which were processed from their 5′ terminus. Correspondingly, deletion of B. subtilis nrnA led to accumulation of RNAs between 2 and 4 nucleotides in length in cellular extracts. Together, these data suggest that many Firmicutes NrnA-like proteins are likely to resemble B. subtilis NrnA to act as a housekeeping enzyme for processing of RNAs between 2 and 4 nucleotides in length.

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Section: Methodsmentioning

confidence: 99%

NrnA is a 5′-3′ exonuclease that processes short RNA substrates in vivo and in vitro

Weiss

Myers

et al. 2022

Nucleic Acids Research

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Tuning the Innate Immune Response to Cyclic Dinucleotides by Using Atomic Mutagenesis

Fin

Rovira

et al. 2020

ChemBioChem

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Cyclic dinucleotides (CDNs) trigger the innate immune response in eukaryotic cells through the stimulator of interferon genes (STING) signaling pathway. To decipher this complex cellular process, a better correlation between structure and downstream function is required. Herein, we report the design and immunostimulatory effect of a novel group of c‐di‐GMP analogues. By employing an “atomic mutagenesis” strategy, changing one atom at a time, a class of gradually modified CDNs was prepared. These c‐di‐GMP analogues induce type‐I interferon (IFN) production, with some being more potent than c‐di‐GMP, their native archetype. This study demonstrates that CDN analogues bearing modified nucleobases are able to tune the innate immune response in eukaryotic cells.

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Chemical Synthesis of the Fluorescent, Cyclic Dinucleotides c^thGAMP

et al. 2022

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The cGAS‐STING pathway is known for its role in sensing cytosolic DNA introduced by a viral infection, bacterial invasion or tumorigenesis. Free DNA is recognized by the cyclic GMP‐AMP synthase (cGAS) catalyzing the production of 2’,3’‐cyclic guanosine monophosphate‐adenosine monophosphate (2’,3’‐cGAMP) in mammals. This cyclic dinucleotide acts as a second messenger, activating the stimulator of interferon genes (STING) that finally triggers the transcription of interferon genes and inflammatory cytokines. Due to the therapeutic potential of this pathway, both the production and the detection of cGAMP via fluorescent moieties for assay development is of great importance. Here, we introduce the paralleled synthetic access to the intrinsically fluorescent, cyclic dinucleotides 2’3’‐cthGAMP and 3’3’‐cthGAMP based on phosphoramidite and phosphate chemistry, adaptable for large scale synthesis. We examine their binding properties to murine and human STING and confirm biological activity including interferon induction by 2’3’‐cthGAMP in THP‐1 monocytes. Two‐photon imaging revealed successful cellular uptake of 2’3’‐cthGAMP in THP‐1 cells.

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Fluorescent analogs of cyclic and linear dinucleotides as phosphodiesterase and oligoribonuclease activity probes

Abstract: 2-Aminopurine or etheno adenosine cyclic dinucleotide probes can report the activity of cyclic dinucleotide PDEs or oligoribonucleases.

Cited by 11 publications

References 58 publications

NrnA is a 5′-3′ exonuclease that processes short RNA substrates in vivo and in vitro

NrnA is a 5′-3′ exonuclease that processes short RNA substrates in vivo and in vitro

Tuning the Innate Immune Response to Cyclic Dinucleotides by Using Atomic Mutagenesis

Chemical Synthesis of the Fluorescent, Cyclic Dinucleotides c^thGAMP

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Fluorescent analogs of cyclic and linear dinucleotides as phosphodiesterase and oligoribonuclease activity probes

Abstract: 2-Aminopurine or etheno adenosine cyclic dinucleotide probes can report the activity of cyclic dinucleotide PDEs or oligoribonucleases.

Cited by 11 publications

References 58 publications

NrnA is a 5′-3′ exonuclease that processes short RNA substrates in vivo and in vitro

NrnA is a 5′-3′ exonuclease that processes short RNA substrates in vivo and in vitro

Tuning the Innate Immune Response to Cyclic Dinucleotides by Using Atomic Mutagenesis

Chemical Synthesis of the Fluorescent, Cyclic Dinucleotides cthGAMP

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Chemical Synthesis of the Fluorescent, Cyclic Dinucleotides c^thGAMP