The architecture of the 10‐23 <scp>DNA</scp>zyme and its implications for <scp>DNA</scp>‐mediated catalysis

Borggräfe, Jan; Gertzen, Christoph G. W.; Viegas, Aldino; Gohlke, Holger; Etzkorn, Manuel

doi:10.1111/febs.16698

Cited by 9 publications

(6 citation statements)

References 51 publications

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“…As a result, the flexibility of dT4 may be reduced, thus preventing the optimal positioning of Me 2+ in the catalytic site. The above observations are in good agreement with Borggräfe et al, who mentioned in their NMR studies that T4 undergoes an Mg 2+ -induced flip-out that may act as a molecular switch during activation [ 7 ].…”

Section: Discussionsupporting

confidence: 90%

“…In this study, the initial geometry of Dz10-23 was obtained by modifying the structure of 7PDU.pdb [ 7 ]; i.e., the cytidine at position 5 of the catalytic loop was replaced by 2′deoxyadenosine (denoted as wt-Dz), while the other oligonucleotides were left unmodified. Additionally, the negative charge of the phosphate group was quenched by sodium ions.…”

Section: Methodsmentioning

confidence: 99%

“…Two of them are assembled by nucleotides composing the catalytic loop, i.e., nucleotides 1–6 at the 5′ end of the loop and nucleotides 12–15 at the 3′ end of the loop. Interestingly, positions 5 and 15 contain 2′-deoxyadenosine (dA), highlighting the importance of this deoxynucleoside for optimal activity [ 6 , 7 ]. Dz10-23 possess significant therapeutic potential due to their ability to cleave RNA involved in the pathogenesis of various diseases such as viral RNA [ 8 ], the mRNA of disease-related proteins [ 9 , 10 ] or regulatory non-coding RNA [ 11 ].…”

Section: Introductionmentioning

confidence: 99%

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The Effect of 8,5′-Cyclo 2′-deoxyadenosine on the Activity of 10-23 DNAzyme: Experimental and Theoretical Study

Cieślak,

Karwowski

2024

IJMS

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The in vivo effectiveness of DNAzymes 10-23 (Dz10-23) is limited due to the low concentration of divalent cations. Modifications of the catalytic loop are being sought to increase the activity of Dz10-23 in physiological conditions. We investigated the effect of 5′S or 5′R 5′,8-cyclo-2′deoxyadenosine (cdA) on the activity of Dz10-23. The activity of Dz10-23 was measured in a cleavage assay using radiolabeled RNA. The Density Functional Tight Binding methodology with the self-consistent redistribution of Mulliken charge modification was used to explain different activities of DNAzymes. The substitution of 2′-deoxyadenosine with cdA in the catalytic loop decreased the activity of DNAzymes. Inhibition was dependent on the position of cdA and its absolute configuration. The order of activity of DNAzymes was as follows: wt-Dz > ScdA5-Dz ≈ RcdA15-Dz ≈ ScdA15-Dz > RcdA5-Dz. Theoretical studies revealed that the distance between phosphate groups at position 5 in RcdA5-Dz was significantly increased compared to wt-Dz, while the distance between O4 of dT4 and nonbonding oxygen of PO2 attached to 3′O of dG2 was much shorter. The strong inhibitory effect of RcdA5 may result from hampering the flexibility of the catalytic loop (increased rigidity), which is required for the proper positioning of Me2+ and optimal activity.

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Section: Discussionsupporting

confidence: 90%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The Effect of 8,5′-Cyclo 2′-deoxyadenosine on the Activity of 10-23 DNAzyme: Experimental and Theoretical Study

Cieślak,

Karwowski

2024

IJMS

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“…Those roles identified by 2D hyperfine spectroscopy agree with a fitting of EPR/ESR binding isotherms recently proposed for the manganese ions [8,35] and with the few cooperatively binding modes proposed for the DNAzymes. [36][37][38][39] The cooperative binding is combined with the intrinsic flexibility of the c4s4 (46mer) architecture.…”

Section: Supporting Informationsupporting

confidence: 88%

Cleaving DNA with DNA: Cooperative Tuning of Structure and Reactivity Driven by Copper Ions

Dantu,

Khalil,

Bria

et al. 2024

Advanced Science

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A copper‐dependent self‐cleaving DNA (DNAzyme or deoyxyribozyme) previously isolated by in vitro selection has been analyzed by a combination of Molecular Dynamics (MD) simulations and advanced Electron Paramagnetic Resonance (Electron Spin Resonance) EPR/ESR spectroscopy, providing insights on the structural and mechanistic features of the cleavage reaction. The modeled 46‐nucleotide deoxyribozyme in MD simulations forms duplex and triplex sub‐structures that flank a highly conserved catalytic core. The DNA self‐cleaving construct can also form a bimolecular complex that has a distinct substrate and enzyme domains. The highly dynamic structure combined with an oxidative site‐specific cleavage of the substrate are two key‐aspects to elucidate. By combining EPR/ESR spectroscopy with selectively isotopically labeled nucleotides it has been possible to overcome the major drawback related to the “metal‐soup” scenario, also known as “super‐stoichiometric” ratios of cofactors versus substrate, conventionally required for the DNA cleavage reaction within those nucleic acids‐based enzymes. The focus on the endogenous paramagnetic center (Cu2+) here described paves the way for analysis on mixtures where several different cofactors are involved. Furthermore, the insertion of cleavage reaction within more complex architectures is now a realistic perspective towards the applicability of EPR/ESR spectroscopic studies.

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“…The experiments revealed that RNA cleavage in BDD with HEG substitutions was 2 times lower than that of BDD versions with inosine substitutions. This difference can be explained by the contribution of the nucleotides of the catalytic core to RNA binding by Dz, a phenomenon reported recently ( 23 , 38 ). At the same time, the relative cleavage activity of HEG-core BDDs and Dz remained similar to that of inosine BDDs and Dz (Figure 9B , 3 and 4; Supplementary Figure S9 ).…”

Section: Resultsmentioning

confidence: 72%

Multivalent DNAzyme agents for cleaving folded RNA

Dubovichenko,

Batsa,

Bobkov

et al. 2024

Nucleic Acids Research

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Multivalent recognition and binding of biological molecules is a natural phenomenon that increases the binding stability (avidity) without decreasing the recognition specificity. In this study, we took advantage of this phenomenon to increase the efficiency and maintain high specificity of RNA cleavage by DNAzymes (Dz). We designed a series of DNA constructs containing two Dz agents, named here bivalent Dz devices (BDD). One BDD increased the cleavage efficiency of a folded RNA fragment up to 17-fold in comparison with the Dz of a conventional design. Such an increase was achieved due to both the improved RNA binding and the increased probability of RNA cleavage by the two catalytic cores. By moderating the degree of Dz agent association in BDD, we achieved excellent selectivity in differentiating single-base mismatched RNA, while maintaining relatively high cleavage rates. Furthermore, a trivalent Dz demonstrated an even greater efficiency than the BDD in cleaving folded RNA. The data suggests that the cooperative action of several RNA-cleaving units can significantly improve the efficiency and maintain high specificity of RNA cleavage, which is important for the development of Dz-based gene knockdown agents.

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The architecture of the 10‐23 DNAzyme and its implications for DNA‐mediated catalysis

Cited by 9 publications

References 51 publications

The Effect of 8,5′-Cyclo 2′-deoxyadenosine on the Activity of 10-23 DNAzyme: Experimental and Theoretical Study

The Effect of 8,5′-Cyclo 2′-deoxyadenosine on the Activity of 10-23 DNAzyme: Experimental and Theoretical Study

Cleaving DNA with DNA: Cooperative Tuning of Structure and Reactivity Driven by Copper Ions

Multivalent DNAzyme agents for cleaving folded RNA

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