Locked nucleoside analogues expand the potential of DNAzymes to cleave structured RNA targets

Birte Vester,Stephen Douthwaite,Lykke H Hansen,B Ravindra Babu,Jesper Wengel,Lars Bo Lundberg,Mads D Sørensen

doi:10.1186/1471-2199-7-19

Birte Vester, Stephen Douthwaite + Show 5 more

Open Access

https://doi.org/10.1186/1471-2199-7-19

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Abstract

BackgroundDNAzymes cleave at predetermined sequences within RNA. A prerequisite for cleavage is that the DNAzyme can gain access to its target, and thus the DNAzyme must be capable of unfolding higher-order structures that are present in the RNA substrate. However, in many cases the RNA target sequence is hidden in a region that is too tightly structured to be accessed under physiological conditions by DNAzymes.ResultsWe investigated how incorporation of LNA (locked nucleic acid) monomers into DNAzymes improves their ability to gain access and cleave at highly-structured RNA targets. The binding arms of DNAzymes were varied in length and were substituted with up to three LNA and α-L-LNA monomers (forming LNAzymes). For one DNAzyme, the overall cleavage reaction proceeded fifty times faster after incorporation of two α-L-LNA monomers per binding arm (kobs increased from 0.014 min-1 to 0.78 min-1).ConclusionThe data demonstrate how hydrolytic performance can be enhanced by design of LNAzymes, and indicate that there are optimal lengths for the binding arms and for the number of modified LNA monomers.

Highlights

DNAzymes cleave at predetermined sequences within RNA
RNA substrates for cleavage RNA substrates contain two consecutive sequences that are complementary to the binding arms of the DNAzymes and bracket the cleavage site between a purine and a pyrimidine (Fig. 1A)
The cleavage sites studied here are displayed in RNA substrates that are based on sequences from the Escherichia coli 23S ribosomal RNA, and possess a range of structural complexity (Fig. 2)

Summary

Introduction

DNAzymes cleave at predetermined sequences within RNA. A prerequisite for cleavage is that the DNAzyme can gain access to its target, and the DNAzyme must be capable of unfolding higher-order structures that are present in the RNA substrate. The discovery that RNA-hydrolytic properties could be encoded within a DNA oligonucleotide indicated potential biotechnological applications in gene silencing These applications might even surpass those of other oligonucleotide-based gene silencing approaches, such as antisense and RNAi technologies, that require the complicity of the cell's own nuclease systems in order to cleave RNA. The use of DNAzymes has been restricted by several limitations, some of which are shared with the other oligonucleotide-based technologies For any of these approaches to be of value, the oligonucleotide must be capable of transversing the cellular membrane and avoid being inactivated by cellular nucleases long enough to find the appropriate cellular compartment where it can bind and induce cleavage at the target (page number not for citation purposes). Modification of DNAzymes to improve their stability against cellular nucleases and their ability to bind and cleave RNA molecules would go a long way towards increasing their general applicability

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