Theranostic DNAzymes

Abstract

DNAzymes are catalytically active DNA molecules that are obtained via in vitro selection. RNA-cleaving DNAzymes have attracted significant attention for both therapeutic and diagnostic applications due to their excellent programmability, stability, and activity. They can be designed to cleave a specific mRNA to down-regulate gene expression. At the same time, DNAzymes can sense a broad range of analytes. By combining these two functions, theranostic DNAzymes are obtained. This review summarizes the progress of DNAzyme for theranostic applications. First, in vitro selection of DNAzymes is briefly introduced, and some representative DNAzymes related to biological applications are summarized. Then, the applications of DNAzyme for RNA cleaving are reviewed. DNAzymes have been used to cleave RNA for treating various diseases, such as viral infection, cancer, inflammation and atherosclerosis. Several formulations have entered clinical trials. Next, the use of DNAzymes for detecting metal ions, small molecules and nucleic acids related to disease diagnosis is summarized. Finally, the theranostic applications of DNAzyme are reviewed. The challenges to be addressed include poor DNAzyme activity under biological conditions, mRNA accessibility, delivery, and quantification of gene expression. Possible solutions to overcome these challenges are discussed, and future directions of the field are speculated.

Keywords: DNAzymes; RNA; biosensors; delivery.; metal ions.

Figure 1

A cartoon showing specific mRNA recognition and cleavage by a DNAzyme. The red square in the mRNA denotes for the targeted cleavage site. The DNAzyme can be directed to the cleavage site by designing the substrate binding arms using Watson-Crick base pairing, thus inhibiting gene expression. The general mechanism of the RNA cleavage reaction is also presented, where the 2′-OH group attacks the scissile phosphate to initiate the cleavage reaction.

Figure 2

A basic scheme of in vitro selection. The rA denotes ribo-adenine and the circled B denotes biotin moiety. The sequences that can cleave the rA bond in the presence of the added metal ions is harvested and amplified by PCR. The biotin is needed for library immobilization so that the negative strand after PCR can be washed away to generate the single-stranded DNA library. In addition, it allows the separation of the cleaved DNA from the rest of the library.

Figure 3

The secondary structures of a few representative RNA-cleaving DNAzymes. (A) 10-23; (B) 8-17; (C) Bipartite II. These three DNAzymes are able to cleave all-RNA substrates, which can be used as therapeutic agents for intracellular RNA cleavage. The (D) NaA43; (E) Ce13d; and (F) EtNa DNAzymes can only cleave a single RNA embedded DNA substrate, which is mainly for sensing applications. The arrowheads denote cleavage site. The “rA” denotes the single ribo-adenosine linkage. The identical nucleotides in NaA43 and Ce13d catalytic core are highlighted in red. These DNAzymes can all use or interact with physiologically important metal ions including Mg²⁺, Ca²⁺, and Na⁺.

Figure 4

Schematic illustration of various oligonucleotide-directed gene therapeutic strategies, including anti-sense oligonucleotides, siRNA and ribozymes/DNAzymes , . In each case, target specificity is achieved by hybridization, but the cleavage mechanisms are different.

Figure 5

(A) A typical sensor design for DNAzyme signaling. F and Q denote for fluorophore and quencher, respectively. (B) A UO₂²⁺ sensor design by adsorption the DNAzyme on AuNPs. AuNPs are potent fluorescence quenchers and also help cell uptake.

Figure 6

(A) A schematic illustration of the RNA-cleaving fluorescent DNAzyme for CEM detection. (B) Converting DNAzyme activity into pH change for colorimetric sensing by immobilizing DNAzyme on a magnetic bead (MB) and extending the enzyme strand to hybridize with a urease bearing DNA.

Figure 7

(A) A design of allosteric DNAzymes with activity modulated by the DNA effector. (B) A scheme showing the design of the MNAzyme to form an active catalytic core. (C) Combining the MNAzyme design with a strand displacement reaction to design enzyme-free signal amplification sensor for DNA detection. Reprinted with the permission from ref. . Copyright 2011 American Chemical Society.

Figure 8

(A) A design of aptazyme by incorporating an aptamer sequence in DNAzyme substrate binding arm region. The target binding releases the competitor DNA to facilitate substrate hybridization. (B) Schematic illustration of the DLISA strategy. Reprinted with permission from ref. . Copyright 2015 American Chemical Society. (C) Schematic of method using PGM to quantify DNAzyme activity. The DNAzyme was attached to a streptavidin-modified magnetic bead, and the substrate was extended to hybridize with DNA-invertase conjugates. Reproduced with permission from ref. . Copyright 2011, Nature Publishing Group.

Figure 9

(A) A schematic illustration of AuNP-based nano-flares for mRNA depletion and detection. (B) A direct adaption of nano-flare concept for fabrication of theranostic DNAzyme. (C) A strategy of detection and knockdown mRNA by adsorption fluorophore-labeled DNAzyme on GO. (D) Schematic illustration of SiO₂ mesoporous encapsulated with fluorophore and caped with DNAzyme for stimuli-responsive content release. Reprinted with the permission from ref. . Copyright 2013 American Chemical Society.