Activatable G-quadruplex based catalases for signal transduction in biosensing

Abstract

Discovery of oxidative catalysis with G-quadruplex•hemin constructs prompted a range of exciting developments in the field of biosensor design. Thus, G-quadruplex based DNAzymes with peroxidase activity found a niche as signal transduction modules in a wide range of analytical applications. The ability of nucleic acid scaffolds to recognise a variety of practically meaningful markers and to translate the recognition events into conformational changes powers numerous sensor design possibilities. In this work, we establish a catalase activity of G-quadruplex•hemin scaffolds. Catalase activated hydrogen peroxide decomposition generates molecular oxygen that forms bubbles. Observation of bubbles is a truly equipment free signal readout platform that is highly desirable in limited resources or do-it-yourself environments. We take a preliminary insight into a G-quadruplex structure-folding topology-catalase activity correlation and establish efficient operating conditions. Further, we demonstrate the platform's potential as a signal transduction modality for reporting on biomolecular recognition using an oligonucleotide as a proof-of-concept target. Ultimately, activatable catalases based on G-quadruplex•hemin scaffolds promise to become valuable contributors towards accessible molecular diagnostics applications.

Scheme 1.

Simplified catalytic cycles of peroxidases and catalases. ‘RS’ = reducing substrate. Iron (III) is blue, iron (IV) is brown.

Figure 1.

Bathochromic shift and increase in intensity of hemin's Soret band indicates hemin's de-aggregation upon interacting with G-quadruplex V. Data for other quadruplexes indicate to a similar trend (Supplementary Figure S2). Hemin concentration is 1 μM, GV concentration is 500 μM in PBS buffer at pH 7.50.

Figure 2.

Representative images (A) and summary (B) of bubbles formation in presence of quadruplexes GI–GVII. Samples were prepared in PBS (pH 7.5) and contained equilibrated G-quadruplexes at indicated levels, hemin at 1 μM, and 29.3% of hydrogen peroxide. Control consisted of all the components except a G-quadruplex. Images in (A) are views from above on 20-ml scintillation vials taken with a cell phone camera. Observations in (B) are taken 30 min after adding hydrogen peroxide. One ‘bubble’ symbol in (B) corresponds to 1–3 bubbles observed, two to 4–8 bubbles, three to 8–12 bubbles, four to 12–18 bubbles, five to almost all the surface area covered (more than 18), dash corresponds to zero (0) bubbles.

Figure 3.

(A) Activation of catalase activity of quadruplexes GVIII and GIX in presence of an oligonucleotide target requires splitting guanine-rich sequences in two parts. We evaluate two different split patterns: a symmetric (6:6) and an asymmetric (9:3). (B) Results over different split and target configurations. Each sample in PBS buffer (pH 7.50) consisted of two quadruplex ‘arms’ (R and L) and target (all at 500 nM concentrations), hemin at 1 μM, and hydrogen peroxide at 29.3%. Control consisted of all the components except an oligonucleotide target. Observations were taken 30 min after adding hydrogen peroxide. One ‘bubble’ symbol in the table correspond to 1–3 bubbles observed, two to 4–8 bubbles, three to 8–12 bubbles. (C) Representative image of response to the target T1 (left) and T2 (right) against a control (no target, centre) taken with 4× objective using Keyance microscope. The samples were prepared in a 96-well plate. The total sample volume was 300 μl. Additionally, conventional cell phone images for GV ‘9:3’ splits are included in Figure S6.