DNAzyme switches for molecular computation and signal amplificationBiosensors and Bioelectronics

About

Authors
Simon M. Bone, Nicole E. Lima, Alison V. Todd
Year
2015
DOI
10.1016/j.bios.2015.03.057
Subject
Electrochemistry / Biophysics / Biotechnology / Biomedical Engineering

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ut a,

Signal amplification r s rid es o n b orte ctiv he put an amplification of signal, which confers significant potential for future biosensing applications where

A and to the ming s with 07; Be

Krishn 011). U selection process were Breaker and Joyce in 1994 (Breaker and mains one of the fastest DNAzymes evolved to date with kcat of been linked with ; Ellington, 1999; atalytic activities e presence of a r, for example, a arious switching approaches have been applied, such as the incorporation of the blocking portion) resulting in the opening of the hairpin and inContents lists available at ScienceDirect .e

Biosensors and

Biosensors and Bioelectronics 70 (2015) 330–337http://dx.doi.org/10.1016/j.bios.2015.03.057dicating presence of the analyte (Deborggraeve et al., 2013; Tian and Mao, 2005; Zhao et al., 2013). Alternatively, the substratebinding arms of a DNAzyme have been truncated with the target then responsible for facilitating the hybridization between the 0956-5663/& 2015 Elsevier B.V. All rights reserved. n Corresponding author at: SpeeDx Pty Ltd, Eveleigh, NSW 2015, Australia.

Fax: þ61 2 9209 4170.

E-mail address: simonb@speedx.com.au (S.M. Bone).Joyce, 1994). The RNA-cleaving 10–23 DNAzyme in particular, reenzyme within a hairpin loop structure. The enzyme is activated by hybridization of the target to one side of the hairpin stem (thediscovery of ‘Ribozymes’; RNA molecules capable of catalyzing reactions such as self-splicing (Lakin et al., 2011), hydrolysis (Lakin et al., 2012) and RNA cleavage (Xing et al., 2011). Following this, the DNA-equivalent ‘DNAzymes’ were generated in the laboratory through in vitro selection where large pools of random DNA sequences were screened for catalytic activity (extensively reviewed in (Silverman, 2005, 2009; Silverman and Begley, 2007)). The pioneers for generating DNAzyme molecules through the in vitro for detection of changes to pH (Elbaz et al., 2010 2011). Many DNAzymes and Ribozymes have also aptamers to create ‘aptazymes’ (Achenbach, 2004

Famulok et al., 2007; Navani and Li, 2006). The c of the aptazymes are then dependent upon th target analyte specific for that particular aptame protein or small molecule.

For the detection of nucleic acid analytes, vacids were not traditionally considered ‘active’ molecules that could catalyze biological reactions, however this changed with the

E6 and the peroxidase-mimicking DNAzymes have both been modified via the inclusion of an ‘i-motif’ (C-rich sequence) to allow b; Shimron et al.,Isothermal 1. Introduction

The ability of nucleic acids (DN predictable manner has given rise ‘nanomachines’ capable of perfor computation and logical operation enzymes (Bath and Turberfield, 20 2006; Chen and Ellington, 2010;

Seeman, 2010; Zhang and Seelig, 2detection of low quantities of target biomarkers is required. & 2015 Elsevier B.V. All rights reserved.

RNA) to interact in a existence of functional movement, molecular out the aid of protein issenhirtz and Willner, an and Simmel, 2011; nlike proteins, nucleic 10 min1 and catalytic efficiency (kcat /KM) of approximately 109 M1 min1 (Santoro and Joyce, 1997, 1998).

Several DNAzymes and Ribozymes have since been further engineered via the addition or deletion of sequence, or by splitting sequences at particular regions to form new secondary structures, all with the purpose of creating ‘molecular switches’. In this case, the activity of the enzyme can be allosterically-regulated by the presence or absence of a particular target analyte or by changes to the external environment. For example, the nucleic acid-cleavingMolecular switch

OligonucleotideDNAzyme switches for molecular comp amplification

Simon M. Bone a,b,n, Nicole E. Lima a, Alison V. Todd a SpeeDx Pty Ltd, Eveleigh, NSW 2015, Australia b The University of New South Wales, Kensington, NSW 2052, Australia a r t i c l e i n f o

Article history:

Received 2 February 2015

Received in revised form 21 March 2015

Accepted 23 March 2015

Available online 31 March 2015

Keywords:

DNAzyme

MNAzyme a b s t r a c t

We have created molecula porarily inactivated by hyb offers significant advantag cleaving enzyme which ca allows for their use as rep operations. Secondly, the a an output and this allows t switch functions as the in target can produce more th journal homepage: wwwation and signal b witches that consist of nucleic-acid cleaving DNAzymes which are temization with blocking oligonucleotides. The unique design of the switches ver existing methods. Firstly, the switches are activated by a nucleic acide made to function only in the presence of a specific target analyte. This r elements which can be easily adapted for use in computational logical ation of each switch produces an active nucleic acid-cleaving DNAzyme as switches to be modularly coupled to one another so that the output of one of another. In addition, the switches are scalable, so that a single input one active DNAzyme output. These features therefore create the means for lsevier.com/locate/bios

Bioelectronics to c ic co

S.M. Bone et al. / Biosensors and Bioelectronics 70 (2015) 330–337 331Scheme 1. Temporary inactivation of DNAzymes via Blocking oligonucleotides (BL)

DNAzyme, comprising two substrate-binding arms (A and B) which flank a catalytDNAzyme and substrate (Sun et al., 2010). Both approaches have since been superseded by recent methods which split the enzyme into smaller sub-parts by separating the catalytic core (Elbaz et al., 2010a; Kolpashchikov, 2007; Mokany et al., 2010). For example, we previously reported the creation of Multi-component Nucleic Acid enzymes (MNAzymes) (Mokany et al., 2010). Here the conserved catalytic core of DNAzymes are split into two parts, and nucleic acid target-sensing arms added to each half-enzyme, now referred to as ‘Partzymes’ (Mokany et al., 2010). Thus, only in the presence of the specific target are the partzymes able to bind adjacently on the target, re-uniting the catalytic core.