G-quadruplex-containing DNAzymes and aptamers are widely applied in many research fields because of their high stability and prominent activities versus the protein counterparts. In this work, G-quadruplex DNAs were equipped with photolabile groups to construct photocaged DNAzymes and aptamers. We incorporated TEEP–OH (thioether-enol phosphate, phenol substituted) into phosphodiester backbone of G-quadruplex DNA by a facile post-synthetic method to achieve efficient photocaging of their activities. Upon light irradiation, the peroxidase-mimicking activity of the caged G-quadruplex DNAzyme was activated, through the transformation of TEEP–OH into a native DNA phosphodiester without any artificial scar. Similarly, the caged G-quadruplex thrombin-binding aptamer also showed light-induced activation of thrombin inhibition activity. This method could serve as a general strategy to prepare photocaged G-quadruplex DNA with other activities for noninvasive control of their functions.

Conventional fluorophores usually undergo aggregation-caused quenching (ACQ), which limits the loading amount of these fluorophores in nanoparticles for bright fluorescence imaging. On the contrary, fluorophores with aggregation-induced emission (AIE) characteristics are strongly fluorescent in their aggregate states and have been an ideal platform for developing highly fluorescent nanomaterials, such as fluorescent silica nanoparticles (FSNPs). In this work, AIE luminogens based on salicylaldehyde hydrazones were embedded in silica nanoparticles through a facile noncovalent approach, which afforded AIE-FSNPs emitting much brighter fluorescence than that of some commercial fluorescein-doped silica and polystyrene nanoparticles. These AIE-FSNPs displaying multiple fluorescence colors were fabricated by a general method, and they underwent much less fluorescence variation due to environmental pH changes compared with fluorescein-hybridized FSNPs. In addition, a DNA aptamer specific to nucleolin was functionalized on the surface of AIE-FSNPs for targeted cell imaging. Fluorescent microscopy and flow cytometry studies both revealed highly selective fluorescence staining of MCF-7 (a cancer cell line with nucleolin overexpression) over MCF-10A (normal) cells by the aptamer-functionalized AIE-FSNPs. The fluorescence imaging in different color channels was achieved using AIE-FSNPs containing each of the AIE luminogens, as well as photoactivatable fluorescent imaging of target cells by the caged AIE fluorophore.Keywords: aggregation-induced emission; cell imaging; DNA aptamer; photoactivatable fluorescence; silica nanoparticle

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.

Photocaged (photoactivatable) biomolecules are powerful tools for noninvasive control of biochemical activities by light irradiation. DNAzymes (deoxyribozymes) are single-stranded oligonucleotides with a broad range of enzymatic activities. In this work, to construct photocaged DNAzymes, we developed a facile and mild postsynthetic method to incorporate an interesting photolabile modification (thioether-enol phosphate, phenol substituted, TEEP–OH) into readily available phosphorothioate DNA. Upon light irradiation, TEEP–OH transformed into a native DNA phosphodiester, and accordingly the DNAzymes with RNA-cleaving activities were turned “on” from its inactive and caged form. Activation of the TEEP–OH-caged DNAzyme by light was also successful inside live cells.

The 2008 Chinese milk scandal caused by the adulteration of melamine encouraged the public to pay attention to melamine detection in milk products and other food stuffs. To allow simple and rapid detection of melamine, we previously isolated an 88 nt melamine aptamer (called Rd29C33) using the structure-switching SELEX. However, this 88 nt oligonucleotide is costly to synthesize, and may also complicate the rational design of biosensors for melamine detection. To overcome this obstacle, we truncated Rd29C33 at several sites, and a 34 nt Rd29C33-T7 melamine aptamer was finally found to show comparable binding affinity and better selectivity to melamine compared to the original 88 nt Rd29C33. Furthermore, a label-free bioassay method for melamine detection was designed by using Rd29C33-T7 and thiazole orange (TO). The addition of melamine to a mixture of Rd29C33-T7 and TO caused the release of TO from Rd29C33-T7, resulting in a decrease of the fluorescence intensity of the solution. A detection limit of 0.12 μM for melamine was achieved using this label-free method. Good recovery ranging from 82.6% to 97.2% for melamine detection in whole milk samples suggested the promise of this bioassay method for application in monitoring melamine in real food stuffs.

The activity of uracil-DNA glycosylase (UDG), an enzyme in the base excision repair, is detected at a high sensitivity by a DNA substrate containing only one uracil through a label-free fluorescent approach, which is also successfully applied for the measurement of UDG inhibitors.

The activity of uracil-DNA glycosylase (UDG), an enzyme in the base excision repair, is detected at a high sensitivity by a DNA substrate containing only one uracil through a label-free fluorescent approach, which is also successfully applied for the measurement of UDG inhibitors.