Circular DNA and DNA/RNA Hybrid Molecules as Scaffolds for Ricin Inhibitor Design

Sturm, Matthew B.; Roday, Setu; Schramm, Vern L.

doi:10.1021/ja068054h

Cited by 35 publications

(50 citation statements)

References 22 publications

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“…1). The loop structure is maintained by a covalent oxime linker with geometry similar to stem-loop substrates (17). This cyclic oligonucleotide contains 5 phosphodiester bridges that are designed to be resistant to phosphodiesterases.…”

Section: Resultsmentioning

confidence: 99%

“…In addition to hairpin stem-loop structures, RTA hydrolyzes adenine from cyclic GAGA loops that possess a 5Ј-to 3Ј-covalently closed synthetic linker (17,18) (Fig. 1).…”

mentioning

confidence: 99%

“…RTA transition state structures have guided the design and synthesis of potent RIP inhibitors (17,29). Inhibitors for RTA and SAP include 1Ј-aza-sugars with a nonhydrolyzable 9-deazaadenine to mimic the elevated pK a of the leaving group (15).…”

mentioning

confidence: 99%

“…1). RTA is active on mammalian ribosomes at physiological pH but is active on small RNAs and inhibited by transition state analogues only at low pH values (17).…”

mentioning

confidence: 99%

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Transition state analogues in structures of ricin and saporin ribosome-inactivating proteins

Sturm

Almo

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Ricin A-chain (RTA) and saporin-L1 (SAP) catalyze adenosine depurination of 28S rRNA to inhibit protein synthesis and cause cell death. We present the crystal structures of RTA and SAP in complex with transition state analogue inhibitors. These tight-binding inhibitors mimic the sarcin-ricin recognition loop of 28S rRNA and the dissociative ribocation transition state established for RTA catalysis. RTA and SAP share unique purine-binding geometry with quadruple -stacking interactions between adjacent adenine and guanine bases and 2 conserved tyrosines. An arginine at one end of the -stack provides cationic polarization and enhanced leaving group ability to the susceptible adenine. Common features of these ribosome-inactivating proteins include adenine leaving group activation, a remarkable lack of ribocation stabilization, and conserved glutamates as general bases for activation of the H2O nucleophile. Catalytic forces originate primarily from leaving group activation evident in both RTA and SAP in complex with transition state analogues.is the catalytic subunit of ricin, a Centers for Disease Control and Prevention category B bioterrorism agent derived from Ricinus communis seeds. RTA catalyzes the depurination of an invariant adenosine residue, A 4234, within the GA 4234 GA tetraloop motif of the highly conserved sarcin-ricin loop of eukaryotic 28S rRNA (1). Clinical trials have exploited the toxicity of RTA in RTA-antibody constructs to kill leukemia and lymphoma cells (e.g., RTA conjugated to anti-CD22) (2-5). Side effects limit the utility of RTA immunotoxins (6, 7). Targeted inhibitors against ribosome-inactivating proteins (RIPs) could improve immunotoxin cancer therapies by rescuing normal cells following toxin treatment.Saporin-L1 (SAP), a homologue of RTA from Saponaria officinalis (soapwort) leaves, exhibits N-glycohydrolase activities on 80S ribosomes, poly(A) RNA, and other cellular . SAP releases multiple adenines from ribosomes, whereas RTA shows exquisite specificity.Truncated oligonucleotide constructs of the ribosomal sarcinricin loop are RTA and SAP substrates (13-16). In addition to hairpin stem-loop structures, RTA hydrolyzes adenine from cyclic GAGA loops that possess a 5Ј-to 3Ј-covalently closed synthetic linker (17, 18) (Fig. 1).Transition state analysis of RTA-mediated depurinations established that hydrolysis of adenine involves a ribocation intermediate, followed by attack of an activated water. Adenine activation is a major driving force for RTA catalysis (19,20). Efficient catalysis by RTA requires the invariant Glu-177 and Arg-180 residues (21-25). RTA and other RIPs have evolved to become near-perfect catalysts for mammalian ribosomes (21,(26)(27)(28). Here, we use transition state analogues to establish the catalytic site features contributing to this remarkable catalytic activity.RTA transition state structures have guided the design and synthesis of potent RIP inhibitors (17, 29). Inhibitors for RTA and SAP include 1Ј-aza-sugars with a nonhydrolyzable 9-deazaadenine to mimic the...

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Section: Resultsmentioning

confidence: 99%

“…In addition to hairpin stem-loop structures, RTA hydrolyzes adenine from cyclic GAGA loops that possess a 5Ј-to 3Ј-covalently closed synthetic linker (17,18) (Fig. 1).…”

mentioning

confidence: 99%

mentioning

confidence: 99%

“…1). RTA is active on mammalian ribosomes at physiological pH but is active on small RNAs and inhibited by transition state analogues only at low pH values (17).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Transition state analogues in structures of ricin and saporin ribosome-inactivating proteins

Sturm

Almo

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…1e). 68 Oxime circularization was also used for circularization of RNA, although only small circles (tetramers) were prepared. 69 Other circularization procedures involve copper catalyzed azide alkyne cycloaddition (CuAAC) 70,71 (Fig.…”

Section: Chemical Strategiesmentioning

confidence: 99%

In vitro circularization of RNA

Müller

Appel

2016

RNA Biology

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Over the past 2 decades, different types of circular RNAs have been discovered in all kingdoms of life, and apparently, those circular species are more abundant than previously thought. Apart from circRNAs in viroids and viruses, circular transcripts have been discovered in rodents more than 20 y ago and recently have been reported to be abundant in many organisms including humans. Their exact function remains still unknown, although one may expect extensive functional studies to follow the currently dominant research into identification and discovery of circRNA by sophisticated sequencing techniques and bioinformatics. Functional studies require models and as such methods for preparation of circRNA in vitro. Here, we will review current protocols for RNA circularization and discuss future prospects in the field.Abbreviations: AMP, adenosine monophosphate; AppRNA, 5 0 -adenylated RNA; ATP, adenosine triphosphate; bp, base pair; CEV-A, citrus exocortis viroid strain A; circRNA, circular RNA; ciRNA, circular intronic RNA; CuAAC, cupper catalyzed azide alkyne cycloaddition; EDC, ethyl-3-(3 0 -dimethylaminopropyl)-carbodiimide; HIV, human immunodeficiency virus; IEciRNA, intron-exon circular RNA; nt, nucleotide; PIE, permuted intron exon; T4 Dnl, T4 DNA Ligase; T4 Rnl, T4 RNA Ligase

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Ricin: Chemistry, Sources, Exposures, Toxicology and Medical Aspects

Lord

Griffiths

2009

General, Applied and Systems Toxicology

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The chapter covers the origins of ricin toxin, which is present in the seeds of the many cultivars of the castor oil plant. The structure and biogenesis of the dimeric (A and B chain toxin) ricin (RCA 60 ) is described, compared with the related, but less toxic, Ricinus agglutinin (RCA 120 ) and the toxic activity of ricin, the N ‐glycosidase activity of the A chain, is explained. The intoxication process is further described and developed through in vitro and in vivo studies, which then focus on several possible routes of exposure. These include inhalation, that route which has been the primary focus of military‐oriented research through oral and parenteral routes of intoxication; histopathology and symptomatology are described, using evidence from human cases of ricin poisoning, wherever possible. Information is then presented on the development of medical countermeasures against ricin poisoning, including pretreatment approaches (vaccines) and postexposure approaches, which include antitoxins. More novel and currently research‐based studies are considered, including inhibitors of N ‐glycosidase, substrate competitors or approaches to interfere with the binding of the ricin B chain to galactose moieties.

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Circular DNA and DNA/RNA Hybrid Molecules as Scaffolds for Ricin Inhibitor Design

Cited by 35 publications

References 22 publications

Transition state analogues in structures of ricin and saporin ribosome-inactivating proteins

Transition state analogues in structures of ricin and saporin ribosome-inactivating proteins

In vitro circularization of RNA

Ricin: Chemistry, Sources, Exposures, Toxicology and Medical Aspects

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