Surface-immobilized DNAzyme-type biocatalysis Loic Stefan, a Thomas Lavergne, b Nicolas Spinelli, b Eric Defrancq b and David Monchaud * a The structure of the double helix of deoxyribonucleic acid (DNA, also called duplex-DNA) was elucidated sixty years ago by Watson, Crick, Wilkins and Franklin. Since then, DNA has continued to hold a fascination for researchers in diverse fields including medicine and nanobiotechnology. Nature has indeed excelled in diversifying the use of DNA: beyond its canonical role of repository of genetic information, DNA could also act as a nanofactory able to perform some complex catalytic tasks in an enzyme-mimicking manner. The catalytic capability of DNA was termed DNAzyme; in this context, a peculiar DNA structure, a quadruple helix also named quadruplex-DNA, has recently garnered considerable interest since its autonomous catalytic proficiency relies on its higher-order folding that makes it suitable to interact efficiently with hemin, a natural cofactor of many enzymes. Quadruplexes have thus been widely studied for their hemoprotein-like properties, chiefly peroxidase-like activity, i.e., their ability to perform hemin-mediated catalytic oxidation reactions. Recent literature is replete with applications of quadruplex-based peroxidase-mimicking DNAzyme systems. Herein, we take a further leap along the road to biochemical applications, assessing the actual efficiency of catalytic quadruplexes for the detection of picomolar levels of surface-bound analytes in an enzyme-linked immunosorbent (ELISA)-type assay. To this end, we exploit an innovative strategy based on the functionalization of DNA by a multitasking platform named RAFT (for regioselectivity addressable functionalized template), whose versatility enables the grafting of DNA whatever its nature (duplex-DNA, quadruplex-DNA, etc.). We demonstrate that the resulting biotinylated RAFT/quadruplex systems indeed acquire catalytic properties that allow for efficient luminescent detection of picomoles of surface-bound streptavidin. We also highlight some of the pitfalls that have to be faced during optimization, notably demonstrating that highly optimized experimental conditions can make DNA pre-catalysts catalytically competent whatever their secondary structures. Introduction The “RNA world” hypothesis, formally articulated in 1986 by W. Gilbert, posits that a common misconception was to consider that ribonucleic acid (RNA) had an accessory role only in the evolutionary process. 1 RNA is not merely the molecular link between DNA (the repository of the genetic information) and proteins (which catalyze complex tasks). Instead, RNA probably preceded both DNA and enzymes in the evolution, thriving on the challenge of exhibiting both informational and catalytic – so-called ribozyme – properties, the former to hold genetic information and ensure an exact mother-to-daughter trans- mission, the latter to ensure self-replication via enzyme- mimicking copying and self-reproducing processes. Thus, DNA probably appeared aer RNA and was subsequently selected as the privileged recipient of the genetic information thanks to its error-correcting double-stranded nature and intrinsic stability. In the evolution timescale, DNA thus somewhat unfairly over- shadowed RNA. The basis for this conclusion was uncompro- misingly sound, but the question of the catalytic capabilities of DNA was a bit too quietly shelved since less than a decade aer, Breaker & Joyce demonstrated that DNA is not solely holding the genetic information but could be enzymatically procient as well. 2 The enzyme-like activity of DNA – termed deoxyribozyme or DNAzyme – was demonstrated through the study of the RNA- cleaving activity of a DNA strand included in a hybrid DNA/RNA duplex. Later on, the scope of the DNAzyme biocatalysis was further extended by Li & Sen, via the demonstration that a single-stranded DNA can acquire enzymatic activity when it serves as an aptamer for hemin, 3 a known cofactor of many hemoprotein enzymes (including peroxidases). The higher- order folding of this G-rich aptamer, established as a quad- ruplex structure, 4 was found to offer a privileged binding site to hemin (one of its external G-quartets); upon addition of a stoi- chiometric oxidant (H 2 O 2 ), the hemin/quadruplex complex promotes peroxidase-like oxidation reactions, notably the easily a Institut de Chimie Mol´ eculaire, Universit´ e de Bourgogne (ICMUB), CNRS UMR6302, Dijon, France. E-mail: david.monchaud@u-bourgogne.fr; Fax: +33 380 396 117; Tel: +33 380 399 043 b D´ epartement de Chimie Mol´ eculaire, Universit´ e Grenoble Alpes, CNRS UMR5250, Grenoble, France Cite this: Nanoscale, 2014, 6, 2693 Received 8th November 2013 Accepted 11th December 2013 DOI: 10.1039/c3nr05954e www.rsc.org/nanoscale This journal is © The Royal Society of Chemistry 2014 Nanoscale, 2014, 6, 2693–2701 | 2693 Nanoscale PAPER