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[Preprint]. 2023 May 31:rs.3.rs-2252941.
doi: 10.21203/rs.3.rs-2252941/v1.

Structure of a 10-23 Deoxyribozyme Exhibiting a Homodimer Conformation

Evan Cramer  1 , Sarah Starcovic  1 , Rebekah Avey  1 , Ali Kaya, Aaron Robart  1
Affiliations

Structure of a 10-23 Deoxyribozyme Exhibiting a Homodimer Conformation

Evan Cramer et al. Res Sq. .

Update in

Abstract

Deoxyribozymes (DNAzymes) are in vitro evolved DNA sequences capable of catalyzing chemical reactions. The RNA cleaving 10-23 DNAzyme was the first DNAzyme to be evolved and possesses clinical and biotechnical applications as a biosensor and a knockdown agent. DNAzymes do not require the recruitment of other components to cleave RNA and can turnover, thus they have a distinct advantage over other knockdown methods (siRNA, CRISPR, morpholinos). Despite this, a lack of structural and mechanistic information has hindered the optimization and application of the 10-23 DNAzyme. Here, we report a 2.7 Å crystal structure of the RNA cleaving 10-23 DNAzyme in a homodimer conformation. Although proper coordination of the DNAzyme to substrate is observed along with intriguing patterns of bound magnesium ions, the dimer conformation likely does not capture the true catalytic form of the 10-23 DNAzyme.

Keywords: DNA; DNAzyme; Deoxyribozyme; RNA; structure.

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Conflict of interest statement

Declarations Competing Interests All authors agree with the contents of the publication and declare no competing interests.

Figures

Figure 1
Figure 1
10–23 DNAzyme crystallization construct and activity. (A) Secondary structure of the 10–23 crystallization construct. (B) Activity of the 10–23 crystallization construct showcasing inhibition of activity with the 2′-OMe substitution at the cleavage site (green dots indicate position of the FAM label).
Figure 2
Figure 2
The crystal structure of the 10–23 DNAzyme forms a homodimer. (A) Secondary structure depicting base-pair interactions between the DNAzyme catalytic cores that coordinate the homodimer conformation. (B) Overview of the crystal structure with crystallization chaperone Asfv PolX.
Figure 3
Figure 3
In vitro evidence of 10–23 dimerization. (A) Restriction enzyme cleavage of the palindromic sequence withing the 10–23 DNAzyme catalytic core. (B) EMSA revealing dimer formation in the presence of substrate and magnesium. (C) EMSA with a heavier unlabeled snapback DNAzyme which shifts and enriches the dimer band (green dots indicate position of the FAM label).
Figure 4
Figure 4
Organization of the active site. (A) Overview of active site with catalytic core bases in orange and paired catalytic core bases in green and substrate (blue) with electron density (s = 1.25) for consensus site bases. (B) Consensus site bases with metal ions coordinated (silver) labeled M1-M3.
Figure 5
Figure 5
Active site comparisons. (A) active site of 10–23 DNAzyme crystal structure. (B) Proposed base-flip conformational change that aligns the 2’OH to the scissile phosphate for ‘in-line’ attack. (C) Alignment of the 10–23 DNAzyme crystal structure active site (blue) and the NMR 10–23 DNAzyme active site (yellow).
See this image and copyright information in PMC

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