On Facts and Artefacts: The Difficulty to Evaluate an Artificial Nuclease

Pitsch, Stefan; Scheffer, Ute; Hey, Markus; Strick, Andreas; Göbel, Michael

doi:10.1002/hlca.200390316

Cited by 7 publications

(4 citation statements)

References 26 publications

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“…The same aggregation takes place at high concentrations of wild-type GCN4 LZ35 [ Fig. S4 (B,C)] and at high concentrations of catalytically active aminobenzimidazole [41] .…”

Section: Discussionmentioning

confidence: 72%

The Leucine Zipper Domains of the Transcription Factors GCN4 and c-Jun Have Ribonuclease Activity

et al. 2010

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Basic-region leucine zipper (bZIP) proteins are one of the largest transcription factor families that regulate a wide range of cellular functions. Owing to the stability of their coiled coil structure leucine zipper (LZ) domains of bZIP factors are widely employed as dimerization motifs in protein engineering studies. In the course of one such study, the X-ray structure of the retro-version of the LZ moiety of yeast transcriptional activator GCN4 suggested that this retro-LZ may have ribonuclease activity. Here we show that not only the retro-LZ but also the authentic LZ of GCN4 has weak but distinct ribonuclease activity. The observed cleavage of RNA is unspecific, it is not suppressed by the ribonuclease A inhibitor RNasin and involves the breakage of 3′,5′-phosphodiester bonds with formation of 2′,3′-cyclic phosphates as the final products as demonstrated by HPLC/electrospray ionization mass spectrometry. Several mutants of the GCN4 leucine zipper are catalytically inactive, providing important negative controls and unequivocally associating the enzymatic activity with the peptide under study. The leucine zipper moiety of the human factor c-Jun as well as the entire c-Jun protein are also shown to catalyze degradation of RNA. The presented data, which was obtained in the test-tube experiments, adds GCN4 and c-Jun to the pool of proteins with multiple functions (also known as moonlighting proteins). If expressed in vivo, the endoribonuclease activity of these bZIP-containing factors may represent a direct coupling between transcription activation and controlled RNA turnover. As an additional result of this work, the retro-leucine zipper of GCN4 can be added to the list of functional retro-peptides.

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“…The same aggregation takes place at high concentrations of wild-type GCN4 LZ35 [ Fig. S4 (B,C)] and at high concentrations of catalytically active aminobenzimidazole [41] .…”

Section: Discussionmentioning

confidence: 72%

The Leucine Zipper Domains of the Transcription Factors GCN4 and c-Jun Have Ribonuclease Activity

et al. 2010

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“…2a Moreover, when using RNA, great attention should be paid to avoid contamination from natural ribonucleases, which are widely present and may lead to artefacts. 42 The design and development of artificial ribonucleases has been extensively reviewed recently, 2a,e and for this reason, the following sections will deal more with HPNP cleavage, and less with RNA and non-activated esters of nucleosides, for which the reader may refer to the cited reviews. Nonetheless, some selected examples will be discussed in order to outline the modern strategy currently under investigation.…”

Section: Rna and Its Modelsmentioning

confidence: 99%

Zinc(ii) complexes as hydrolytic catalysts of phosphate diester cleavage: from model substrates to nucleic acids

2007

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The development of synthetic agents able to hydrolytically cleave phosphate diester bonds with high efficiency is a fascinating challenge, which will ultimately open the way to artificial nucleases able to compete with the natural enzymes. This Perspective highlights the progress reported in the realization of hydrolytic catalysts based on the Zn 2+ ion, a metal ion which, due to its peculiar properties, is a very promising candidate. The review critically examines the reactivity of such catalysts toward model substrates and nucleic acids, paying particular attention to the strategies that can be pursued to improve efficiency and sequence selectivity.

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“…We reported earlier on the difficulty to rule out false positive effects. 1,5 Substrate specificity (Figure 3) is already a strong argument against contamination. As a further control, we have investigated the cleavage of substrate 10, an oligonucleotide analogous to 9 that consists of two major parts.…”

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confidence: 99%

Site-Specific Cleavage of RNA by a Metal-Free Artificial Nuclease Attached to Antisense Oligonucleotides

et al. 2006

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RNA cleaving tris(2-aminobenzimidazoles) have been attached to DNA oligonucleotides via disulfide or amide bonds. The resulting conjugates are effective organocatalytic nucleases showing substrate and site selectivity as well as saturation kinetics. The benzimidazole conjugates also degrade enantiomeric RNA. This observation rules out contamination effects as an alternative explanation of RNA degradation. The pH dependency shows that the catalyst is most active in the deprotonated state. Typical half-lifes of RNA substrates are in the range of 12-17 h. Thus, conjugates of tris(2-aminobenzimidazoles) can compete with the majority of metal-dependent artificial nucleases.

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On Facts and Artefacts: The Difficulty to Evaluate an Artificial Nuclease

Cited by 7 publications

References 26 publications

The Leucine Zipper Domains of the Transcription Factors GCN4 and c-Jun Have Ribonuclease Activity

The Leucine Zipper Domains of the Transcription Factors GCN4 and c-Jun Have Ribonuclease Activity

Zinc(ii) complexes as hydrolytic catalysts of phosphate diester cleavage: from model substrates to nucleic acids

Site-Specific Cleavage of RNA by a Metal-Free Artificial Nuclease Attached to Antisense Oligonucleotides

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