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Vester, Birte; Hansen, Lykke Haastrup; Lundberg, L.; Babu, B. Ravindra; Sørensen, Mads D.; Wengel, Jesper; Douthwaite, Stephen

doi:10.1186/1471-2199-7-19

Cited by 40 publications

(41 citation statements)

References 34 publications

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“…This result parallels those reported in previous studies on the 10 -23 motif (11,(13)(14)(15)(16), demonstrating that the inclusion of LNA monomers in the target-binding arms is advantageous for deoxyribozymes in general. More fundamentally, our work has addressed the mechanistic basis of the advantages conferred by LNAs, providing a detailed rationale for the effects reported by us and by other authors.…”

Section: Discussionsupporting

confidence: 81%

“…The lower concentration requirements may explain, for exam-5 This conclusion presumably also holds for the 10 -23 constructs described in previous studies (11)(12)(13)(14)(15)(16). The association rates for the 10 -23 deoxyribozyme already approach those observed for short complementary oligonucleotides (k Ϫ1 values Ͼ10 8 min Ϫ1 ) (36, 37), and it seems unlikely that these rates may be substantially increased in LNA-armed constructs.…”

Section: Discussionmentioning

confidence: 58%

“…It is very likely that the 10 -23 LNAzyme constructs described in previous studies (11)(12)(13)(14)(15)(16)(17) behave similar to Lz122. The lower concentration requirements may explain, for exam-5 This conclusion presumably also holds for the 10 -23 constructs described in previous studies (11)(12)(13)(14)(15)(16).…”

Section: Discussionmentioning

confidence: 99%

“…LNA residues are capable of normal Watson-Crick base pairing, and the locked sugar enhances the preorganization of the phosphate backbone, stabilizing the helical (A-type) structure of a duplex (8 -10). Several studies have shown that the inclusion of a few LNA monomers into the arms of the 10 -23 deoxyribozyme significantly improves the performances of this catalyst against small RNA substrates and, more pronouncedly, against long RNA transcripts, promoting cleavage of highly structured RNAs (11)(12)(13)(14)(15)(16)(17).…”

mentioning

confidence: 99%

“…With respect to a minimal kinetic scheme for a deoxyribozyme reaction (Fig. 1B), it remains to be established whether the increased affinity alone can explain the improved kinetics, or if LNA also accelerates the intramolecular cleavage step (described by the kinetic constant k 2 ) (11,16). Furthermore, the LNA monomers could in principle improve the RNA binding affinity of deoxyribozymes via an increased association rate (k 1 ) or a reduced dissociation rate (k Ϫ1 ), or both; data from model systems are somewhat contradictory on this point (18,19).…”

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confidence: 99%

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The Advantages of Being Locked

Donini

Clerici

Wengel

et al. 2007

Journal of Biological Chemistry

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RNA-cleaving deoxyribozymes can be used for the sequencespecific knockdown of mRNAs. It was previously shown that activity of these deoxyribozymes is enhanced when their substrate-binding arms include some locked nucleic acid (LNA) residues, but the mechanistic basis of this enhancement was not explored. Here we dissected the kinetics and thermodynamics underlying the reaction of LNA-containing 8 -17 deoxyribozymes. Four 8 -17 constructs were designed to target sequences within the E6 mRNA from human papillomavirus type 16. When one of these deoxyribozymes (DNAzymes) and the corresponding LNA-armed enzyme (LNAzyme) were tested against a minimal RNA substrate, they showed similar rates of substrate binding and similar rates of intramolecular cleavage, but the LNAzyme released its substrate more slowly. The superior thermodynamic stability of the LNAzyme-substrate complex led to improved performances in reactions carried out at low catalyst concentrations. The four DNAzymes and the corresponding LNAzymes were then tested against extended E6 transcripts (>500 nucleotides long). With these structured substrates, the LNAzymes retained full activity, whereas the DNAzymes cleaved extremely poorly, unless they were allowed to pre-anneal to their targets. These results imply that LNAzymes can easily overcome the kinetic barrier represented by local RNA structure and bind to folded targets with a faster association rate as compared with DNAzymes. Such faster annealing to structured targets can be explained by a model whereby LNA monomers favor the initial hybridization to short stretches of unpaired residues ("nucleation"), which precedes disruption of the local mRNA structure and completion of the binding process.In the past decade, several RNA-cleaving deoxyribozymes (DNAzymes) 4 have been isolated in the laboratory through the application of a combinatorial technique known as "in vitro selection" (1). The two most prominent examples are the 8 -17 and 10 -23 deoxyribozymes, originally identified in the Joyce laboratory (2). Both types of deoxyribozymes comprise a central "core" and two substrate-binding arms that can be varied in length and sequence. The chemical stability of these catalysts, their ease of synthesis, and versatility in the sequence-specific cleavage of RNA have prompted a wealth of studies on the potential use of DNAzymes in therapy, to inhibit the expression of disease-causing genes by selectively binding and cleaving the corresponding mRNAs (e.g. (3-5)).However, several factors limit the practical use of DNAzymes as anti-gene agents. For example, it may be difficult to achieve an intercellular concentration of DNAzyme high enough to ensure stable binding to the target mRNA and hence efficient knockdown. In part, this problem can be countered by lengthening the substrate-recognition arms, thus increasing the affinity of the DNAzyme for its target, but this entails the synthesis of larger DNA molecules, which are less manageable and more prone to the adoption of alternative structures and to off-target ...

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Section: Discussionsupporting

confidence: 81%

Section: Discussionmentioning

confidence: 58%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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The Advantages of Being Locked

Donini

Clerici

Wengel

et al. 2007

Journal of Biological Chemistry

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Locked Nucleic Acids: Properties, Applications, and Perspectives

Nielsen

Wengel

2008

Modified Nucleosides

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A decade has passed since the introduction of locked nucleic acid (LNA) [1][2][3]. This chapter provides a review and a current status on the features and applications of LNA. Attention is focused on the structural chemistry of LNA and on its applications for therapeutics and probes. With regards to the latter point, the chapter is based on former comprehensive reviews on LNA applications [4][5][6] and, for that reason, is focused on results reported subsequently, with no intention of being comprehensive.The field of nucleic acid chemistry has evolved considerably during the past few decades, leading from synthetic developments via molecular recognition into nucleic acid-based therapeutics, powerful diagnostic probes, and modern nanobiotechnology. From the structural perspective, the central issue has been the thorough understanding of the basis for molecular recognition and the selective formation of nucleic acid complexes, initially the classic Watson-Crick-type double helix. The central creative challenge for synthetic chemists has been to design simple chemical perturbations of the natural duplex in order to increase our knowledge within nucleic acid chemical biology, and to bring this knowledge into applied nucleic acids research [7-9].The major motivation behind the design of nucleic acid analogues has, over the past two decades, been the antisense strategy aimed at the silencing of genes by strong binding towards mRNA, with or without associated RNA-cleavage. Two central problems have motivated the chemical effort: (1) the physiological instability of natural oligonucleotides; and (2) the relatively moderate affinity of short natural oligonucleotides for their RNA-targets [10,11]. If a solution to these problems -as well as to the delicate question of cellular delivery -can be found, then oligonucleotides will present unlimited therapeutic perspectives. Among the more than 1000 chemical analogues tested for these properties, a large part seems to induce a high degree of resistance towards nucleolytic degradation, although this is dependent on the sequence composition, the number and positioning of the chemical modifications within the sequence, and on the degree of perturbation compared to the natural chemical structure. On the other hand, significantly increasing the RNA-affinity of an Modified Nucleosides: in Biochemistry, Biotechnology and Medicine. Edited by Piet Herdewijn

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Therapeutic DNAzymes: From Structure Design to Clinical Applications

et al. 2023

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Therapeutic DNAzymes have unceasingly intrigued the scientific community owing to their prosperous gene regulation capability. The efficacy of DNAzymes against many types of diseases has been extensively studied for over two decades. However, the high expectations for DNAzymes are still not translated to the clinic because of their low effectiveness in vivo. Over the last five years, several aspects have been considered to optimize DNAzyme‐integrated therapeutics, including structural stability, mechanism exploration, cell internalization rate, cofactor activation, and off‐target effects. Hence, this review first discusses the early monotherapy and structural design of DNAzymes. Subsequently, the latest modes of action are reviewed, followed by an elaboration on structural stabilization strategies considering the catalytic core and substrate‐binding arms. DNAzyme‐based synergistic therapy is then examined, highlighting responsive carrier construction, synergistic effects, the latest discovered advanced functions, and off‐target concerns. Beyond this, key clinical advances in DNAzyme‐based therapy are elucidated by showcasing clinical progress. Finally, future trends and development challenges for DNAzyme‐powered therapeutics in the coming years are discussed in detail.

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Untitled

Cited by 40 publications

References 34 publications

The Advantages of Being Locked

The Advantages of Being Locked

Locked Nucleic Acids: Properties, Applications, and Perspectives

Therapeutic DNAzymes: From Structure Design to Clinical Applications

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