Cationic Peptide Conjugation Enhances the Activity of Peroxidase-Mimicking DNAzymes

Abstract

Peroxidase-mimicking DNAzymes containing G-quadruplex structures are widely applied in chemistry as catalysts and signal amplification for biosensing. Enhancing the catalytic activity of these DNAzymes can therefore improve the performance of many catalysts and biosensors using them. In this work, we synthesized cationic peptide conjugates of peroxidase-mimicking DNAzymes, which were found to exhibit both enhanced peroxidase and oxidase activities up to 4-fold and 3-fold compared with the original DNAzymes, respectively. Further investigation suggested that the enhanced activity was ascribed to the stabilization of parallel DNA G-quadruplex structures and hemin binding by the cationic peptide covalently attached to the DNAzyme. Such a mechanism of activity enhancement was successfully utilized for biosensing applications with improved sensitivity and broadened target range. Hydrogen peroxide (H2O2) detection in K(+)-free solutions by the DNAzyme-peptide conjugate showed 2-fold sensitivity enhancement over the unmodified DNAzyme under the same condition, and the activity switch by target-induced cleavage of the DNAzyme-peptide conjugate was also used for the detection of caspase 3 protease with enzymatic amplification in homogeneous solutions.

Catalytic Activities of Multimeric G-Quadruplex DNAzymes

“…Similar rate-enhancements were observed by the addition of the nucleotide ATP to DNAzyme reactions [12,13]. It has also been reported that the conjugation of hemin with the G4-quadruplex moiety via covalent linkage [14] or with cationic peptides [15] can enhance the activities of DNAzymes. Other studies have focused on the rate-enhancing effects of flanking adenine or cytosine nucleotides on G-quadruplex activities [16][17][18][19].…”

“…The absorbance intensity at λ = 415 nm (corresponding to the amount of ABTS • formed) was measured as a function of time. The initial rates (V o ) were calculated from the slope of the initial (30 s) linear portion of the increase in absorbance [15,39].…”

A Thermophilic Tetramolecular G‐Quadruplex/Hemin DNAzyme

“…This intermediate finally withdraws one electron from another ABTS molecule to provide the initial porph⋅Fe III cofactor. Upon addition of H 2 O 2 , the dynamics of the absorbance change at 404 nm demonstrated a fast degradation tendency within five minutes (Supporting Information, Figure S11), while G4, G5, and G6 DNAzyme were characterized by the low degradation rates (Figure B). Interestingly, G4A and AG4A DNAzyme showed similar degradation rates, which were higher than those of G4 modified at 5′ and/or 3′ ends with dT or dC.…”

The Effect of Adenine Repeats on G‐quadruplex/hemin Peroxidase Mimicking DNAzyme Activity

“…G-quadruplex/hemin complexesc an catalyze the oxidation of substrates (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonica cid) (ABTS),3 ,3',5,5'-tetramethylbenzidine (TMB), or luminol) in the presence of H 2 O 2 ,a nd the subsequentc olor change (ABTS, l max = 415 nm when oxidized) has been used in colorimetric and/or chemiluminescent detection of various targets.C ompared with proteinous enzymes, G-quadruplex/hemin DNAzymes have many advantages. Smallm olecule additives (ATP or spermine), [13][14][15] may be added to the G-quadruplex/hemin system;a lternatively, covalent conjugation of ac ationicp eptide [16] or hemin to G-quadruplex, [17] or chemical modification of G-quadruplexes [18] have been used. Furthermore, it is asimple and sensitive biosensor and molecular machine that is suitable for the detection of multiple targets such as metal ions, [3] small molecules, [4] and biomolecules (DNA and proteins), [5,6] which were described in recent reviews.…”

“…[7][8][9][10][11][12] To enhance the performance and activity of peroxidase-mimicking DNAzymes, different strategies have been proposed. Smallm olecule additives (ATP or spermine), [13][14][15] may be added to the G-quadruplex/hemin system;a lternatively, covalent conjugation of ac ationicp eptide [16] or hemin to G-quadruplex, [17] or chemical modification of G-quadruplexes [18] have been used. Recently,f lankings equences such as d(CCC) [19] and dA, [20] in intramolecular parallel G-quadruplexes, were found to enhance the activity of G-quadruplex/hemin DNAzymes.…”