Efficient Access to 3′-Terminal Azide-Modified RNA for Inverse Click-Labeling Patterns

Santner, Tobias; Hartl, Markus; Bister, Klaus; Micura, Ronald

doi:10.1021/bc400513z

Cited by 52 publications

(52 citation statements)

References 57 publications

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“…[5] 2′-Azidonucleosides have proven useful for analysis of RNA produced in vitro by chemical synthesis. [6] N6-propargyl as well as C2- and C7-ethynyl adenosine have also been demonstrated to be useful probes for metabolic labeling of transcription and polyadenylation. [7] Despite this progress, a holistic description of the types of analogues that can be utilized to track RNA synthesis and processing inside living cells remains to be systematically interrogated.…”

mentioning

confidence: 99%

Metabolic Incorporation of Azide Functionality into Cellular RNA

Nainar¹,

Beasley²,

Fazio³

et al. 2016

ChemBioChem

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Real-time tracking of RNA expression can provide insight into the mechanisms used to generate cellular diversity, as well as help determine the underlying causes of disease. Here we present the exploration of azide-modified nucleoside analogues and their ability to be metabolically incorporated into cellular RNA. We report robust incorporation of adenosine analogues bearing azide handles at both the 2′- and N6-positions; 5-methylazidouridine was not incorporated into cellular RNA. We further demonstrate selectivity of our adenosine analogues for transcription and polyadenylation. We predict that azidonucleosides will find widespread utility in examining RNA functions inside living cells, as well as in more complex systems such as tissues and living animals.

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confidence: 99%

Metabolic Incorporation of Azide Functionality into Cellular RNA

Nainar¹,

Beasley²,

Fazio³

et al. 2016

ChemBioChem

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“…In recent years CuAAC has been established as another reaction in this standard toolbox (14), with myriad applications ranging from labeling of nucleic acids with dyes for in vivo imaging (15), grafting of oligonucleotides onto electrode surfaces (16, 17), and a novel procedure that exploits copper chelation for stepwise CuAAC of oligonucleotides with an unsymmetrical bis-azido bridging molecule (18). …”

Section: Resultsmentioning

confidence: 99%

Heterogeneous Catalysis for Azide-Alkyne Bioconjugation in Solution Via Spin Column: Attachment of Dyes and Saccharides to Peptides and DNA

et al. 2015

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Copper-catalyzed azide-alkyne cycloaddition (CuAAC) "click" chemistry is widely used and has demonstrated particular utility for bio-orthogonal conjugation reactions. Here we describe a one-pot, heterogeneous bioconjugation and purification method for selectively activated CuAAC. A Cu(II) precursor, with either the neutral ligand 1,10-phenanthroline-5,6-dione or the anionic ligand 4,7-diphenyl-1,10-phenanthroline-disulfonic acid, is converted to the active Cu(I) species within an ion-exchange matrix using zinc amalgam as the reducing agent. The Cu(I) complexes are then layered on top of a size-exclusion matrix within a commercial microcentrifuge spin column; passing a mixture of an ethynyl-labeled biomolecule and an azide-bearing ligand through the column results in clean and efficient coupling. The methodology is demonstrated by glycosylating a DNA oligonucleotide as well as by labeling a membrane-penetrating peptide (octa-arginine) with a coumarin dye.

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“…Compound 2 was synthesized and immobilized on GE Healthcare Custom Primer Support™ Amino resin (GE Healthcare cat#17-5214-98) by following the previously reported procedure [38]. The functionalized resin was packed into empty Bioautomation MerMade columns (MM-1000-1) (4.00 mg resin per column).…”

Section: Methodsmentioning

confidence: 99%

“…The RNA oligonucleotide synthesis was performed as described above. After synthesis, the RNA was cleaved from the resin as previously described [38]. After evaporating the resulting solution to dryness, the RNA was resuspended in DMSO (anhydrous, 100 μL).…”

Section: Methodsmentioning

confidence: 99%

Cobalt-based paramagnetic probe to study RNA-protein interactions by NMR

Seebald

DeMott

Ranganathan

et al. 2017

Journal of Inorganic Biochemistry

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Paramagnetic resonance enhancement (PRE) is an NMR technique that allows studying three-dimensional structures of RNA-protein complexes in solution. RNA strands are typically spin labeled using nitroxide reagents, which provide minimal perturbation to the native structure. The current work describes an alternative approach, which is based on a Co2+-based probe that can be covalently attached to RNA in the vicinity of the protein’s binding site using ‘click’ chemistry. Similar to nitroxide spin labels, the transition metal based probe is capable of attenuating NMR signal intensities from protein residues localized <40 Å away. The extent of attenuation is related to the probe’s distance, thus allowing for construction of the protein’s contact surface map. This new paradigm has been applied to study binding of HIV-1 nucleocapsid protein 7, NCp7, to a model RNA pentanucleotide.

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Efficient Access to 3′-Terminal Azide-Modified RNA for Inverse Click-Labeling Patterns

Cited by 52 publications

References 57 publications

Metabolic Incorporation of Azide Functionality into Cellular RNA

Metabolic Incorporation of Azide Functionality into Cellular RNA

Heterogeneous Catalysis for Azide-Alkyne Bioconjugation in Solution Via Spin Column: Attachment of Dyes and Saccharides to Peptides and DNA

Cobalt-based paramagnetic probe to study RNA-protein interactions by NMR

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