Biosensors and Bioelectronics 70 (2015) 330–337
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
DNAzyme switches for molecular computation and signal amplification Simon M. Bone a,b,n, Nicole E. Lima a, Alison V. Todd a,b a b
SpeeDx Pty Ltd, Eveleigh, NSW 2015, Australia The University of New South Wales, Kensington, NSW 2052, Australia
art ic l e i nf o
a b s t r a c t
Article history: Received 2 February 2015 Received in revised form 21 March 2015 Accepted 23 March 2015 Available online 31 March 2015
We have created molecular switches that consist of nucleic-acid cleaving DNAzymes which are temporarily inactivated by hybridization with blocking oligonucleotides. The unique design of the switches offers significant advantages over existing methods. Firstly, the switches are activated by a nucleic acidcleaving enzyme which can be made to function only in the presence of a specific target analyte. This allows for their use as reporter elements which can be easily adapted for use in computational logical operations. Secondly, the activation of each switch produces an active nucleic acid-cleaving DNAzyme as an output and this allows the switches to be modularly coupled to one another so that the output of one switch functions as the input of another. In addition, the switches are scalable, so that a single input target can produce more than one active DNAzyme output. These features therefore create the means for amplification of signal, which confers significant potential for future biosensing applications where detection of low quantities of target biomarkers is required. & 2015 Elsevier B.V. All rights reserved.
Keywords: DNAzyme MNAzyme Signal amplification Molecular switch Oligonucleotide Isothermal
1. Introduction The ability of nucleic acids (DNA and RNA) to interact in a predictable manner has given rise to the existence of functional ‘nanomachines’ capable of performing movement, molecular computation and logical operations without the aid of protein enzymes (Bath and Turberfield, 2007; Beissenhirtz and Willner, 2006; Chen and Ellington, 2010; Krishnan and Simmel, 2011; Seeman, 2010; Zhang and Seelig, 2011). Unlike proteins, nucleic acids were not traditionally considered ‘active’ molecules that could catalyze biological reactions, however this changed with the discovery 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 selection process were Breaker and Joyce in 1994 (Breaker and Joyce, 1994). The RNA-cleaving 10–23 DNAzyme in particular, remains one of the fastest DNAzymes evolved to date with kcat of n Corresponding author at: SpeeDx Pty Ltd, Eveleigh, NSW 2015, Australia. Fax: þ61 2 9209 4170. E-mail address: [email protected] (S.M. Bone).
http://dx.doi.org/10.1016/j.bios.2015.03.057 0956-5663/& 2015 Elsevier B.V. All rights reserved.
10 min 1 and catalytic efficiency (kcat /KM) of approximately 109 M 1 min 1 (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-cleaving E6 and the peroxidase-mimicking DNAzymes have both been modified via the inclusion of an ‘i-motif’ (C-rich sequence) to allow for detection of changes to pH (Elbaz et al., 2010b; Shimron et al., 2011). Many DNAzymes and Ribozymes have also been linked with aptamers to create ‘aptazymes’ (Achenbach, 2004; Ellington, 1999; Famulok et al., 2007; Navani and Li, 2006). The catalytic activities of the aptazymes are then dependent upon the presence of a target analyte specific for that particular aptamer, for example, a protein or small molecule. For the detection of nucleic acid analytes, various switching approaches have been applied, such as the incorporation of the enzyme within a hairpin loop structure. The enzyme is activated by hybridization of the target to one side of the hairpin stem (the blocking portion) resulting in the opening of the hairpin and indicating 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
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Scheme 1. Temporary inactivation of DNAzymes via Blocking oligonucleotides (BL) to create molecular switches. The switches are composed of a nucleic acid-modifying DNAzyme, comprising two substrate-binding arms (A and B) which flank a catalytic core region, C. This DNAzyme is then hybridized to a BL comprising two inhibitory regions (A′ and B′) which flank an intermediate region, D. The BL can either be designed to hybridize with the DNAzyme in a traditional manner (denoted BL1), such that its 5′ and 3′ termini hybridize with the 3′ and 5′ termini of the DNAzyme. This results in the formation of a blocked duplexed DNAzyme structure (top right). Alternatively, when the 5′ and 3′ ends of the BL (denoted BL2) are paired with the 5′ and 3′ ends of the DNAzyme, a Quasi-circular DNAzyme structure is formed between the two oligonucleotides. In either conformation, the DNAzyme is blocked and temporarily inactive.
DNAzyme 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. The ability to switch catalytic nucleic acid enzymes between inactive and active states exemplifies their dynamic nature and broad utility. However, the successful outputs from such modifications may generally be limited to proof-of-concept biosensing applications with targets in high abundance. Therefore, without additional amplification systems in place, these biosensors may lack the sensitivity required for detection of targets from clinical samples. Towards this goal, we previously reported the development of a DNA-only cascade which functioned to amplify signal following the detection of a range of different target analytes (Bone et al., 2014). Herein, we have extended this work by expanding the methods for switching the catalytic activity of DNAzymes between on and off states, which we then utilize for both molecular computation and signal amplification purposes. The switches described create the means for a DNAzyme to be initially inactivated (Scheme 1) and subsequently ‘switched on’ in the presence of a target nucleic acid sequence (Scheme 2). Following this, they are modulated further such that they can be used to calculate OR and AND logical operations from either multiple input targets to produce the same output, or from a single input
target to produce more than one output. Finally, the activation of DNAzymes in this manner provides a unique opportunity to switch on additional DNAzymes and we outline different methods of amplifying signal via the construction of both cross-catalytic and auto-catalytic feedback cascades.
2. Experimental 2.1. Materials and reagents All synthetic oligonucleotides were purchased from either Integrated DNA Technologies (Coralville, IA, USA) or Biosearch Technologies (Petaluma, CA, USA). The oligonucleotide sequences are listed in Table S-1, Supplementary Information. The PCR buffer II and MgCl2 were purchased from Life Technologies. 2.2. Isothermal reaction method Reactions contained a master mix consisting of 50 nM each of partzyme A and partzyme B, 200 nM of fluorescent substrate and 1x PCR buffer II. Reactions also contained one or more switch structures comprising a DNAzyme and blocking oligonucleotide (BL) (specified for each individual method) and may have also have contained a synthetic nucleic acid target or no target (no target control reaction). Reactions were initiated by the addition of 45 mM of MgCl2 with a 25 mL final volume. Reactions were incubated at a constant temperature (specified for each individual method) in a CFX96™ Real-Time PCR Detection System (Bio-Rad) with fluorescence measured every 30 s. All samples were run in either duplicate or triplicate. Unless presented otherwise, results
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Scheme 2. MNAzymes are utilized to re-activate the catalytic activity of DNAzymes in the presence of a target nucleic acid. The MNAzyme (red) hybridizes to the target (green) and cleaves the MNAzyme substrate (also red), present as part of the intermediate region within the BL of each switch structure (Blocked duplexed DNAzyme or Quasi-circular DNAzyme). This results in the truncation of the BL into two smaller fragments and the subsequent release and activation of the DNAzyme (blue) where it can then cleave a fluorescently-labelled substrate to produce a fluorescent signal. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
in figures are the average of replicates and were plotted using Microsoft Excel (Version 14). 2.3. MNAzyme activation of DNAzyme switch structures The reactions reported in Fig. 1 panel A, used an MNAzyme comprising 20 nM of Partzyme A (TFRCA4/3-P) and 20 nM of Partzyme B (TFRCB5/3-P), which hybridize adjacently to 10 nM of the target nucleic acid (AF-TFRC). This sequence is homologous to the human Transferrin receptor (TFRC) gene (Genbank: NC_000003.11). The active MNAzyme cleaved blocked duplexed DNAzyme structures consisting of 20 nM of a BL (refer to Table S-1, Supplementary Data) and 10 nM of DNAzyme (Dz2(9:8)) with cleavage designed to activate the DNAzyme. The reactions reported in Fig. 1 panel B, used an MNAzyme comprising 20 nM of Partzyme A (TFRCA4/6- P) and 20 nM of Partzyme B (TFRCB5/6- P) which also hybridize adjacently to 10 nM of the target nucleic acid (AF-TFRC). The active MNAzyme cleaved quasi-circular DNAzyme structures consisting of 20 nM of a BL (refer to Table S-1, Supplementary Data) and 10 nM of DNAzyme (Dz2(9:8)) with cleavage designed to activate the DNAzyme. For both the blocked duplexed and quasi-circular DNAzyme switches, the active DNAzyme produced signal by cleaving 200 nM of the fluorescently-labelled DNAzyme substrate (Sub2-FIB). All reactions were performed according to the Isothermal reaction method with a 45 °C reaction temperature for 40 minutes. Signal to background differences were calculated by subtracting the average final fluorescence (arbitrary units) of the background (no target) reactions from the averaged final fluorescence of reactions containing the target. These values were plotted as bar graphs using Microsoft Excel (Version 14).
2.4. DNAzyme switches for molecular logic The OR logic gate reaction reported in Fig. 2 panel A, consisted of a quasi-circle comprising 10 nM of a DNAzyme (Dz77_55(9:9)) and 15 nM of BL (C(R31c)). The BL comprised two adjacent substrates (Mz1 substrate and Mz2 substrate) within its intermediate region. The Mz1 substrate was cleaved by MNAzyme 1 (Mz1) consisting of 50 nM Partzyme A (cfb2A4/72) and 50 nM Partzyme B (cfb2B5/72), with either no target, 1 nM or 0.5 nM of Target 1 (Af-cfb2). Target 1 is homologous to sequence from the Streptococcus agalactiae Christie-Atkins-Munch-Petersen (cAMP) gene (GenBank: X72754.1). The Mz2 substrate was cleaved by MNAzyme 2 (Mz2) consisting of 50 nM Partzyme A (ctrA5A4/56-P) and 50 nM Partzyme B (ctrA5B5/56-P), with either no target, 1 nM or 0.5 nM of Target 2 (AF-ctrA4). Target 2 is homologous to sequence from the Neisseria meningitidis capsular transport (ctrA) gene (GenBank: HQ156899.1). Following its release from the quasi-circle, the active DNAzyme cleaved 200 nM of the fluorescently-labelled DNAzyme substrate (Sub77_55-FIB). Reactions were performed following the Isothermal reaction method, with a 54 °C reaction temperature for 150 min. The YES logic gate reaction reported in Fig. 2 panel B, consisted of a quasi-circle comprising both 100 nM DzA (Dz2(8:7)) and 100 nM DzB (Dz6(8:7)) each designed to hybridize with 200 nM BLA (C4Sub45(22:23)) and 200 nM BLB (C4Sub45T(21:24)). In addition, reactions contained an MNAzyme consisting of 100 nM Partzyme A (cfb2A4/45- P) and 100 nM Partzyme B (cfb2B5/45-P), and either no target or 20 nM of Target 1. Following their release from the quasi-circle, the active Dz1 and Dz2 cleaved 200 nM of the fluorescently-labelled Dz1 substrate (Sub2-FIB) and the fluorescently-labelled Dz2 substrate (Sub6-TRB2) respectively.
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oligonucleotides (BL)
Fig. 1. Normalized signal to background ratios for four different design variations of each DNAzyme switch. Detailed sequence designs and raw fluorescence data are shown in the Supplementary Data (Figs. S-1 and S-2 respectively). (A) Blocked duplexed DNAzymes. Design 1 (purple) – 3 nt unbound, Design 2 (red) – 3 nt unbound with G–T wobble pairs, Design 3 (blue) – 5 nt unbound, Design 4 (green) – 5 nt unbound with G–T wobble pairs. (B) Quasi-circular DNAzymes. Design 1 (purple) – 3 nt unbound with G–T wobbles, Design 2 (red) – 6 nt unbound, Design 3 (blue) – 8 nt unbound, Design 4 (green) – 12 nt unbound. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Reactions were performed following the Isothermal reaction method, with a 48 °C reaction temperature for 150 min. 2.5. Signal amplification cascades All reactions reported in Fig. 3 consisted of the MNAzyme (Mz1), comprising 50 nM of Partzyme A (cfb2A4/72) and 50 nM of Partzyme B (cfb2B5/72) and either no target, 100 pM or 20 pM of Target 1 (AF-cfb2). The ‘Circle A only’ reaction in Fig. 3 panel A contained Circle A; comprising 10 nM of the Dz1 (Dz77_55(8:9)) and 19 nM of the BL (C(R43d)). The ‘cross-catalytic cascade’ reaction in Fig. 3 panel B contained Circle A as well as Circle B; comprising 10 nM of Dz2 (Dz45(8:9)R) and 14 nM of the BL (C(R36c)). The ‘auto-catalytic cascade’ reaction in Fig. 3 panel C contained Circle C; comprising 8.5 nM of Dz1 and 10 nM of the BL (C(R22h)). In all reactions reported in Fig. 3, fluorescent signal was generated by Dz1 which, when released from the relevant quasi-circle, cleaved 200 nM of the fluorescently-labelled Dz1 substrate (Sub77_55-FIB). Reactions were performed following the Isothermal reaction method, with a 54 °C reaction temperature for 150 min. 3. Results and discussion 3.1. Creation of DNAzyme molecular switches with blocking
The DNAzyme switches reported here comprise two partially complementary oligonucleotides; a DNAzyme capable of cleaving a nucleic acid substrate and a Blocking oligonucleotide (BL) (Scheme 1). The BL consists of two terminal inhibitory regions (A′ and B′), which are each complementary to a portion of the DNAzyme, and a third intermediate region, D, which does not hybridize to the DNAzyme. The intermediate region is universal and can, if desired, consist of any sequence that can be recognized and/or be modified by another molecule such as an enzyme. The BL can be designed so that it hybridizes with the DNAzyme in one of two different ways. Firstly, the 5′ and 3′ termini of the BL can be designed to hybridize with the 3′ and 5′ termini of the DNAzyme in a traditional manner to form a blocked duplexed DNAzyme structure (BL1). Alternatively, the 5′ and 3′ termini of the BL can be designed to hybridize with the 5′ and 3′ termini of the DNAzyme (BL2). In order to maintain correct Watson–Crick base pairing however, a quasi-circular structure is formed. In either conformation, the DNAzyme is initially temporarily inactive and unable to modify any substrates. In this study, an MNAzyme was utilized to recognize a target nucleic acid and activate the switches by cleaving the substrate within the intermediate region (Scheme 2). When this substrate is cleaved, it results in the truncation of the BL into two shorter fragments which can no longer hybridize efficiently to the DNAzyme under the same isothermal reaction conditions. Consequently, the DNAzyme is released and its catalytic activity is restored. The active DNAzyme can then cleave its nucleic acid substrate, which is dual-labelled with a fluorophore and quencher moiety, such that cleavage results in the production of a detectable fluorescent signal. For each of the two types of DNAzyme switches (Blocked duplexed DNAzymes or Quasi-circular DNAzymes), four different design variations (labelled Designs 1–4) were created. The detailed sequence designs are provided in Fig. S-1, Supplementary Data. For the blocked duplexed DNAzyme switches (Fig. S-1, panel A), Designs 1 and 2 contain all but three nucleotides of the DNAzyme sequence hybridized to the BL. These bases are located at approximately the center of the catalytic core. Designs 3 and 4 are similar to Designs 1 and 2, but instead contain five un-paired catalytic core nucleotides. Additionally, both Designs 2 and 4 contain two G-T wobble base pairs located approximately half-way within the center of each inhibitory BL region and the DNAzyme. Thus, each of the four designs are unique. The raw fluorescence data produced from reactions containing each design are shown in Fig. S-2 panel A, Supplementary Data. This data was normalized so that background signal (no target) was subtracted from the target signal and is presented in Fig. 1, panel A. The greatest signal-tobackground difference was produced from Design 2. This design provides an optimal balance with a higher melting temperature (Tm) at the center of the duplex to help keep the DNAzyme blocked in the absence of target, but with the inclusion of the additional G-T wobble base pairs to help reduce the total Tm slightly, increasing the ability of the two oligonucleotides to separate following cleavage of the BL by the MNAzyme. Design 4 did not have as great a signal to background difference as Design 2 because the combination of the G–T wobble base pairs with five un-paired bases lead to a greater instability between the two oligonucleotides under the assay conditions. Designs 1 and 3 on the other hand, were too stable under these conditions and therefore less favorable due to a lack of separation of the DNAzyme from the cleaved BL. For the quasi-circular DNAzyme switches (Fig. S-1, panel B), Designs 1–4 contain all but three, six, eight and twelve nucleotides of the DNAzyme sequence hybridized to the BL respectively. These
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Fig. 2. Quasi-circular DNAzyme switches can be utilized for molecular logic gates. (A) (i) An OR logic gate consisting of a quasi-circle comprising two adjacent substrates for Mz1 and Mz2. The presence of Target 1 or Target 2 results in the activation of Mz1 or Mz2 respectively. Cleavage of either Mz1 or Mz2 substrates by the corresponding MNAzyme results in DNAzyme activation from the switch as evidenced by the FAM output signal summarized in the truth table (ii) and shown in the raw data plot (iii). A comparable increase in fluorescence is observed with 1 nM of Targets 1 or 2, or 0.5 nM of each. (B) (i) A YES gate consists of a quasi-circular DNAzyme structure comprising two different BL (BLA and BLB) and two different DNAzymes (DzA and DzB), Cleavage of the MNAzyme substrate present within both BLA and BLB results in the simultaneous release and activation of both DzA and DzB. The active DNAzymes can then cleave their substrates which are fluorescently-labelled with FAM and Texas Red (TxR) fluorophores respectively. This results in an increase in both FAM and TxR output signals, as summarized in the truth table (ii) and shown in the raw data plot (iii).
un-paired bases are also located at approximately the center of the catalytic core. Additionally, Design 1 contains two G–T wobble base pairs located approximately half-way between the center of each inhibitory BL region and the DNAzyme. The raw fluorescence data produced from reactions with these designs is shown in Fig. S-2 panel B, Supplementary Data. The normalized signal is presented in Fig. 1, panel B. Here, the greatest signal to background difference was produced from Design 2 containing six un-paired catalytic core nucleotides. This was followed by Designs 1 and 3 which were not as favorable due to a higher level of background signal in each case. Design 4 with the lowest Tm, was highly nonspecific with very minimal difference between signal and background observed. The blocked duplexed and quasi-circular switches outlined here are unique for two reasons. Firstly, they temporarily inhibit a nucleic acid-modifying DNAzyme and secondly, in order to reactivate this DNAzyme they require the cleavage activity of another nucleic acid modifying enzyme (e.g. a DNAzyme or MNAzyme). Thus, the output of one switch can be used as the input of
another. This provides a significant advantage over existing methods in that they can be used to create circular feedback cascades (Section 3.3). In contrast, many existing methods utilize peroxidase-mimicking DNAzymes (Elbaz et al., 2009; Guo et al., 2010; Qiu et al., 2010; Zhu et al., 2011) which have not yet been demonstrated to catalyze modification of nucleic acid substrates. Additionally, other methods outlined to date may block nucleic acid-modifying DNAzymes with complementary oligonucleotides, however the DNAzyme is typically released the via a strand-displacing mechanism whereby the target simply hybridizes to the complementary (blocking) strand and does not cleave it (Deborggraeve et al., 2013; Kahan-Hanum et al., 2013; Orbach et al., 2012; Tian and Mao 2005; Wang et al., 2002). The two DNAzyme switches outlined here proved to be successful provided an optimal Tm existed between the two oligonucleotides for the reaction temperature. Theoretically, a higher reaction temperature would favor a stronger Tm and vice versa for a lower reaction temperature. This is of course within the limits of the enzymes temperature range for optimal catalytic activity. At the time of
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Fig. 3. Amplification of signal is achieved via the construction of cross-catalytic and auto-catalytic feedback cascades. (A) Circle A only. (B) A cross-catalytic cascade between Circles A and B. (C) An auto-catalytic cascade produced by Circle C. In each scenario, the MNAzyme (red) recognizes a target (green) and cleaves the Mz1 substrate (also red) which is present un-labelled within the intermediate region of each quasi-circular structure. Fluorescent signal is produced when Dz1 (blue) is released from a quasi-circle, where it then cleaves a fluorescently-labelled Dz1 substrate (also blue). The raw fluorescence data for each scenario is shown underneath the schematic, with signals shown from 100 pM of target (green), 20 pM of target (blue) and the no target control (black). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
development, the quasi-circular switches were more advanced and hence were used for further manipulations as described in Sections 3.2 and 3.3. 3.2. DNAzyme switches for molecular computational logic The ability to activate the DNAzyme switches in the presence of a specific target analyte and provide a detectable output also creates the potential for their use in molecular computation. Molecular computing elements hold great interest due to their potential to be exploited for logical control of biological processes (Orbach et al., 2012). Such systems may be used to function as “programmable drugs”, responding only in the presence of abnormal conditions (Elbaz et al., 2010a; Kahan-Hanum et al., 2013; Willner et al., 2012). To demonstrate potential for computation, we modified a quasi-circular DNAzyme switch (corresponding to Design 2, Fig. S-1, panel B) in one of two ways. In the first scenario (Fig. 2, panel A), we incorporated two adjacent substrate sequences within the intermediate region of the BL. Each substrate can be cleaved by a different MNAzyme recognizing a different target nucleic acid. MNAzyme 1 (Mz1) therefore recognizes Target 1 and cleaves the Mz1 substrate. Similarly, MNAzyme 2 (Mz2) recognizes Target 2 and cleaves the Mz2 substrate. An OR logic gate was constructed, whereby cleavage of at least one of the substrates present within the BL was all that was required for the release of the DNAzyme from the quasi-circle and the subsequent restoration of its catalytic activity. There was a comparable
increase in fluorescence signal when different targets were either individually provided or provided together at an equivalent concentration (the final concentration was 1 nM in each case). This indicates that cleavage at only one location within the intermediate region of the BL is all that is required for DNAzyme release. The inclusion of multiple adjacent substrates within the BL of the quasi-circular switch to create the OR logic gate is also advantageous for the creation of a circular feedback cascade (shown in Section 3.3). This is because the quasi-circle BL is designed to temporarily inactivate a DNAzyme and the addition of an initiator catalytic nucleic acid enzyme e.g. an MNAzyme that recognizes and cleaves the same substrate as the DNAzyme could be problematic since the initiator enzyme itself could compete with the DNAzyme for hybridization to the complementary regions of the BL. The two adjacent substrates therefore separate the MNAzyme initiation step from the subsequent DNAzyme catalysis and therefore minimize any unwanted binding. In the second scenario (Fig. 2, panel B), a unique quasi-circle was constructed containing two different DNAzyme molecules. In this case, a YES logic gate occurs with two possible outputs; i.e. both DNAzymes can be activated following cleavage of one of two BL molecules by an MNAzyme responsive to a single nucleic acid target (Target 1). The two different DNAzymes (DzA and DzB) were each hybridized to distinct terminal regions of the two BL molecules (BLA and BLB). Each BL contained the same MNAzyme substrate within its intermediate region. Cleavage of this substrate by
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the MNAzyme resulted in the release of DzA and DzB, which were capable of recognizing and cleaving the DzA and DzB substrates respectively. Here we chose to label these substrates with two different fluorophores; FAM for DzA substrate and Texas Red (TxR) for DzB substrate, so that the release of each DNAzyme from the quasi-circle was individually monitored by observing the fluorescence signal from each channel. When the target is present, an increase in fluorescence is observed above that of the no target control in both the FAM and TxR channels, indicating that both DzA and DzB are released from the switch. This therefore demonstrates that the quasi-circular switch can be scaled-up to incorporate multiple DNAzymes which can be activated by a single target. Such a switch may potentially increase the speed of a feedback cascade as the number of active DNAzymes would theoretically double with each activation event. 3.3. DNAzyme switches as components of signal amplification cascades In addition to logical operations, the DNAzyme switches developed herein can also be used as the basic components for signal amplification cascades. To demonstrate this, low picomolar concentrations of nucleic acid target were provided for an MNAzyme (Mz1), which can cleave an un-labelled substrate (Mz1 substrate) present within the intermediate region of the BL of a quasi-circular DNAzyme structure. Three different scenarios are presented in Fig. 3. In all three scenarios, a DNAzyme (Dz1) is hybridized to a BL. Fluorescent signal is produced when Dz1 is released from its BL and is then able to cleave a fluorescently-labelled Dz1 substrate. The first scenario (Fig. 3, panel A) consists of only a single quasicircle ‘Circle A’, which comprises a BL containing the substrate for Mz1 and an adjacent substrate for another DNAzyme (Dz2) which as such is not amenable to cleavage by activated DNAzyme Dz1. When Mz1 cleaves the Mz1 substrate, Dz1 is released and can only cleave the fluorescently-labelled Dz1 substrate, generating signal. Thus there is no potential for additional DNAzymes to be activated. Consequently, in this scenario, there was only a slow and linear increase in fluorescence signal with 100 pM target and no detectable increase in signal for 20 pM of target above the no target control. In the second scenario (Fig. 3, panel B), a cross-catalytic cascade between Circle A and a different quasi-circle, ‘Circle B’ was constructed as a means to promote amplification of signal. In this instance, Dz1 when released from Circle A, also has the potential to cleave an un-labelled Dz1 substrate present within the intermediate region of the BL of Circle B and vice versa for the DNAzyme (Dz2) of Circle B. Each time this occurs, additional Dz1 and Dz2 molecules are released and their catalytic activity restored. In this scenario, the signal for 100 pM of target increased at a faster rate and reached a plateau much sooner than the corresponding non-cascade reaction in Fig. 3 panel A. The 20 pM target signal was also now detectable above the no target control. Alternatively, the third scenario involved the construction of an auto-catalytic cascade containing only one quasi-circle ‘Circle C’ (Fig. 3 panel C)). In this instance, the DNAzyme that was initially hybridized to the terminal regions of the BL (Dz1), also had the potential to bind and cleave a substrate within the intermediate region of the same BL molecule. The auto-catalytic quasi-circle also contained the substrate for Mz1, thus initiating the cascade in a target-dependent manner. The auto-catalytic cascade provided by Circle C also resulted in fluorescent signals for 100 pM and 20 pM target concentrations which reached a plateau much sooner than that of the corresponding non-cascade reaction in Fig. 3 panel A. These signals were both also faster than the cross-catalytic cascade shown in Fig. 3 panel B. The auto-catalytic ability of the quasicircle switch was primarily achieved by its unique design in that
there is greater complementarity between the DNAzyme and the 5′ and 3′ ends of the BL (regions A′ and B′ from Scheme 1) rather than the internal substrate sequence with which it also shares complementarity (region D from Scheme 1). This therefore means that in addition to the substrate-binding arms, part of the DNAzyme core sequence is also designed to hybridize with the noncleaved BL. The advantage to creating an auto-catalytic system over that of a cross-catalytic system was that the reaction components were effectively halved and there was no unwanted complementarity between different DNAzymes and BL molecules such as that which could occur in a cross-catalytic cascade. This allowed us to use lower BL to DNAzyme ratios, making reactions proceed faster. We have therefore demonstrated that the DNAzyme switches outlined here can be successfully incorporated into circular feedback cascades for the purpose of signal amplification. This is primarily due to their unique design requiring a nucleic acid-cleaving input and producing a nucleic acid-cleaving DNAzyme as an output. Thus, each individual switch possesses the ability to feed into one another creating a molecular ‘domino effect’. Previous switching attempts have largely utilized peroxidase-mimicking DNAzymes as the output, however, despite the broad applications of peroxidase-mimicking DNAzymes, these enzymes cannot catalyze the modification of nucleic acid substrates, thus they provide no mechanism for activation of additional catalytic nucleic acids capable of mediating signal amplification. The cascades produced here have advantages due to their ability to be responsive to different types of analytes (e.g. nucleic acids, small molecules, metal ions) (Bone et al., 2014). Additional advantages of the approach including the capacity for single nucleotide discrimination and multiplexing were demonstrated by our recent use of the autocatalytic quasi-circle switch in a ‘DNA-only cascade’ (Bone et al., 2014). From that study, the detection limit for nucleic acid targets was calculated to be 2.3 pM, which is comparable to other DNAzyme-based and protein enzyme-free methods reported, but provides a significant improvement in detection speed as many of these methods require much longer incubation times of up to 12 h (Bone et al., 2014). In addition, our method can be performed within a single-tube, with a single-step protocol. Future improvements to sensitivity are hypothesized to involve further optimization of the switch design and the oligonucleotide concentrations.
4. Conclusion This paper outlines the construction of nucleic acid-modifying DNAzyme switches. The DNAzymes were temporarily inactivated via Watson–Crick hybridization to complementary regions of blocking oligonucleotides and restoration of DNAzyme catalytic activity was then achieved via modification of an intermediate region between the two DNAzyme complementary regions of the BL, which only occurred in the presence of a specific target analyte. The switches can be easily modified for the purposes of both molecular computation and for constructing circular feedback cascades in order to achieve signal amplification, conferring significant potential for biosensing applications.
Acknowledgements This work was supported by SpeeDx Pty Ltd. The authors would like to acknowledge Dr. Elisa Mokany and Nicole Hasick for their technical assistance.
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Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.03.057.
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Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
DNAzyme switches for molecular computation and signal amplification Simon M. Bone a,b,n, Nicole E. Lima a, Alison V. Todd a,b a b
SpeeDx Pty Ltd, Eveleigh, NSW 2015, Australia The University of New South Wales, Kensington, NSW 2052, Australia
art ic l e i nf o
a b s t r a c t
Article history: Received 2 February 2015 Received in revised form 21 March 2015 Accepted 23 March 2015 Available online 31 March 2015
We have created molecular switches that consist of nucleic-acid cleaving DNAzymes which are temporarily inactivated by hybridization with blocking oligonucleotides. The unique design of the switches offers significant advantages over existing methods. Firstly, the switches are activated by a nucleic acidcleaving enzyme which can be made to function only in the presence of a specific target analyte. This allows for their use as reporter elements which can be easily adapted for use in computational logical operations. Secondly, the activation of each switch produces an active nucleic acid-cleaving DNAzyme as an output and this allows the switches to be modularly coupled to one another so that the output of one switch functions as the input of another. In addition, the switches are scalable, so that a single input target can produce more than one active DNAzyme output. These features therefore create the means for amplification of signal, which confers significant potential for future biosensing applications where detection of low quantities of target biomarkers is required. & 2015 Elsevier B.V. All rights reserved.
Keywords: DNAzyme MNAzyme Signal amplification Molecular switch Oligonucleotide Isothermal
1. Introduction The ability of nucleic acids (DNA and RNA) to interact in a predictable manner has given rise to the existence of functional ‘nanomachines’ capable of performing movement, molecular computation and logical operations without the aid of protein enzymes (Bath and Turberfield, 2007; Beissenhirtz and Willner, 2006; Chen and Ellington, 2010; Krishnan and Simmel, 2011; Seeman, 2010; Zhang and Seelig, 2011). Unlike proteins, nucleic acids were not traditionally considered ‘active’ molecules that could catalyze biological reactions, however this changed with the discovery 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 selection process were Breaker and Joyce in 1994 (Breaker and Joyce, 1994). The RNA-cleaving 10–23 DNAzyme in particular, remains one of the fastest DNAzymes evolved to date with kcat of n Corresponding author at: SpeeDx Pty Ltd, Eveleigh, NSW 2015, Australia. Fax: þ61 2 9209 4170. E-mail address: [email protected] (S.M. Bone).
http://dx.doi.org/10.1016/j.bios.2015.03.057 0956-5663/& 2015 Elsevier B.V. All rights reserved.
10 min 1 and catalytic efficiency (kcat /KM) of approximately 109 M 1 min 1 (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-cleaving E6 and the peroxidase-mimicking DNAzymes have both been modified via the inclusion of an ‘i-motif’ (C-rich sequence) to allow for detection of changes to pH (Elbaz et al., 2010b; Shimron et al., 2011). Many DNAzymes and Ribozymes have also been linked with aptamers to create ‘aptazymes’ (Achenbach, 2004; Ellington, 1999; Famulok et al., 2007; Navani and Li, 2006). The catalytic activities of the aptazymes are then dependent upon the presence of a target analyte specific for that particular aptamer, for example, a protein or small molecule. For the detection of nucleic acid analytes, various switching approaches have been applied, such as the incorporation of the enzyme within a hairpin loop structure. The enzyme is activated by hybridization of the target to one side of the hairpin stem (the blocking portion) resulting in the opening of the hairpin and indicating 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
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Scheme 1. Temporary inactivation of DNAzymes via Blocking oligonucleotides (BL) to create molecular switches. The switches are composed of a nucleic acid-modifying DNAzyme, comprising two substrate-binding arms (A and B) which flank a catalytic core region, C. This DNAzyme is then hybridized to a BL comprising two inhibitory regions (A′ and B′) which flank an intermediate region, D. The BL can either be designed to hybridize with the DNAzyme in a traditional manner (denoted BL1), such that its 5′ and 3′ termini hybridize with the 3′ and 5′ termini of the DNAzyme. This results in the formation of a blocked duplexed DNAzyme structure (top right). Alternatively, when the 5′ and 3′ ends of the BL (denoted BL2) are paired with the 5′ and 3′ ends of the DNAzyme, a Quasi-circular DNAzyme structure is formed between the two oligonucleotides. In either conformation, the DNAzyme is blocked and temporarily inactive.
DNAzyme 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. The ability to switch catalytic nucleic acid enzymes between inactive and active states exemplifies their dynamic nature and broad utility. However, the successful outputs from such modifications may generally be limited to proof-of-concept biosensing applications with targets in high abundance. Therefore, without additional amplification systems in place, these biosensors may lack the sensitivity required for detection of targets from clinical samples. Towards this goal, we previously reported the development of a DNA-only cascade which functioned to amplify signal following the detection of a range of different target analytes (Bone et al., 2014). Herein, we have extended this work by expanding the methods for switching the catalytic activity of DNAzymes between on and off states, which we then utilize for both molecular computation and signal amplification purposes. The switches described create the means for a DNAzyme to be initially inactivated (Scheme 1) and subsequently ‘switched on’ in the presence of a target nucleic acid sequence (Scheme 2). Following this, they are modulated further such that they can be used to calculate OR and AND logical operations from either multiple input targets to produce the same output, or from a single input
target to produce more than one output. Finally, the activation of DNAzymes in this manner provides a unique opportunity to switch on additional DNAzymes and we outline different methods of amplifying signal via the construction of both cross-catalytic and auto-catalytic feedback cascades.
2. Experimental 2.1. Materials and reagents All synthetic oligonucleotides were purchased from either Integrated DNA Technologies (Coralville, IA, USA) or Biosearch Technologies (Petaluma, CA, USA). The oligonucleotide sequences are listed in Table S-1, Supplementary Information. The PCR buffer II and MgCl2 were purchased from Life Technologies. 2.2. Isothermal reaction method Reactions contained a master mix consisting of 50 nM each of partzyme A and partzyme B, 200 nM of fluorescent substrate and 1x PCR buffer II. Reactions also contained one or more switch structures comprising a DNAzyme and blocking oligonucleotide (BL) (specified for each individual method) and may have also have contained a synthetic nucleic acid target or no target (no target control reaction). Reactions were initiated by the addition of 45 mM of MgCl2 with a 25 mL final volume. Reactions were incubated at a constant temperature (specified for each individual method) in a CFX96™ Real-Time PCR Detection System (Bio-Rad) with fluorescence measured every 30 s. All samples were run in either duplicate or triplicate. Unless presented otherwise, results
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Scheme 2. MNAzymes are utilized to re-activate the catalytic activity of DNAzymes in the presence of a target nucleic acid. The MNAzyme (red) hybridizes to the target (green) and cleaves the MNAzyme substrate (also red), present as part of the intermediate region within the BL of each switch structure (Blocked duplexed DNAzyme or Quasi-circular DNAzyme). This results in the truncation of the BL into two smaller fragments and the subsequent release and activation of the DNAzyme (blue) where it can then cleave a fluorescently-labelled substrate to produce a fluorescent signal. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
in figures are the average of replicates and were plotted using Microsoft Excel (Version 14). 2.3. MNAzyme activation of DNAzyme switch structures The reactions reported in Fig. 1 panel A, used an MNAzyme comprising 20 nM of Partzyme A (TFRCA4/3-P) and 20 nM of Partzyme B (TFRCB5/3-P), which hybridize adjacently to 10 nM of the target nucleic acid (AF-TFRC). This sequence is homologous to the human Transferrin receptor (TFRC) gene (Genbank: NC_000003.11). The active MNAzyme cleaved blocked duplexed DNAzyme structures consisting of 20 nM of a BL (refer to Table S-1, Supplementary Data) and 10 nM of DNAzyme (Dz2(9:8)) with cleavage designed to activate the DNAzyme. The reactions reported in Fig. 1 panel B, used an MNAzyme comprising 20 nM of Partzyme A (TFRCA4/6- P) and 20 nM of Partzyme B (TFRCB5/6- P) which also hybridize adjacently to 10 nM of the target nucleic acid (AF-TFRC). The active MNAzyme cleaved quasi-circular DNAzyme structures consisting of 20 nM of a BL (refer to Table S-1, Supplementary Data) and 10 nM of DNAzyme (Dz2(9:8)) with cleavage designed to activate the DNAzyme. For both the blocked duplexed and quasi-circular DNAzyme switches, the active DNAzyme produced signal by cleaving 200 nM of the fluorescently-labelled DNAzyme substrate (Sub2-FIB). All reactions were performed according to the Isothermal reaction method with a 45 °C reaction temperature for 40 minutes. Signal to background differences were calculated by subtracting the average final fluorescence (arbitrary units) of the background (no target) reactions from the averaged final fluorescence of reactions containing the target. These values were plotted as bar graphs using Microsoft Excel (Version 14).
2.4. DNAzyme switches for molecular logic The OR logic gate reaction reported in Fig. 2 panel A, consisted of a quasi-circle comprising 10 nM of a DNAzyme (Dz77_55(9:9)) and 15 nM of BL (C(R31c)). The BL comprised two adjacent substrates (Mz1 substrate and Mz2 substrate) within its intermediate region. The Mz1 substrate was cleaved by MNAzyme 1 (Mz1) consisting of 50 nM Partzyme A (cfb2A4/72) and 50 nM Partzyme B (cfb2B5/72), with either no target, 1 nM or 0.5 nM of Target 1 (Af-cfb2). Target 1 is homologous to sequence from the Streptococcus agalactiae Christie-Atkins-Munch-Petersen (cAMP) gene (GenBank: X72754.1). The Mz2 substrate was cleaved by MNAzyme 2 (Mz2) consisting of 50 nM Partzyme A (ctrA5A4/56-P) and 50 nM Partzyme B (ctrA5B5/56-P), with either no target, 1 nM or 0.5 nM of Target 2 (AF-ctrA4). Target 2 is homologous to sequence from the Neisseria meningitidis capsular transport (ctrA) gene (GenBank: HQ156899.1). Following its release from the quasi-circle, the active DNAzyme cleaved 200 nM of the fluorescently-labelled DNAzyme substrate (Sub77_55-FIB). Reactions were performed following the Isothermal reaction method, with a 54 °C reaction temperature for 150 min. The YES logic gate reaction reported in Fig. 2 panel B, consisted of a quasi-circle comprising both 100 nM DzA (Dz2(8:7)) and 100 nM DzB (Dz6(8:7)) each designed to hybridize with 200 nM BLA (C4Sub45(22:23)) and 200 nM BLB (C4Sub45T(21:24)). In addition, reactions contained an MNAzyme consisting of 100 nM Partzyme A (cfb2A4/45- P) and 100 nM Partzyme B (cfb2B5/45-P), and either no target or 20 nM of Target 1. Following their release from the quasi-circle, the active Dz1 and Dz2 cleaved 200 nM of the fluorescently-labelled Dz1 substrate (Sub2-FIB) and the fluorescently-labelled Dz2 substrate (Sub6-TRB2) respectively.
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oligonucleotides (BL)
Fig. 1. Normalized signal to background ratios for four different design variations of each DNAzyme switch. Detailed sequence designs and raw fluorescence data are shown in the Supplementary Data (Figs. S-1 and S-2 respectively). (A) Blocked duplexed DNAzymes. Design 1 (purple) – 3 nt unbound, Design 2 (red) – 3 nt unbound with G–T wobble pairs, Design 3 (blue) – 5 nt unbound, Design 4 (green) – 5 nt unbound with G–T wobble pairs. (B) Quasi-circular DNAzymes. Design 1 (purple) – 3 nt unbound with G–T wobbles, Design 2 (red) – 6 nt unbound, Design 3 (blue) – 8 nt unbound, Design 4 (green) – 12 nt unbound. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Reactions were performed following the Isothermal reaction method, with a 48 °C reaction temperature for 150 min. 2.5. Signal amplification cascades All reactions reported in Fig. 3 consisted of the MNAzyme (Mz1), comprising 50 nM of Partzyme A (cfb2A4/72) and 50 nM of Partzyme B (cfb2B5/72) and either no target, 100 pM or 20 pM of Target 1 (AF-cfb2). The ‘Circle A only’ reaction in Fig. 3 panel A contained Circle A; comprising 10 nM of the Dz1 (Dz77_55(8:9)) and 19 nM of the BL (C(R43d)). The ‘cross-catalytic cascade’ reaction in Fig. 3 panel B contained Circle A as well as Circle B; comprising 10 nM of Dz2 (Dz45(8:9)R) and 14 nM of the BL (C(R36c)). The ‘auto-catalytic cascade’ reaction in Fig. 3 panel C contained Circle C; comprising 8.5 nM of Dz1 and 10 nM of the BL (C(R22h)). In all reactions reported in Fig. 3, fluorescent signal was generated by Dz1 which, when released from the relevant quasi-circle, cleaved 200 nM of the fluorescently-labelled Dz1 substrate (Sub77_55-FIB). Reactions were performed following the Isothermal reaction method, with a 54 °C reaction temperature for 150 min. 3. Results and discussion 3.1. Creation of DNAzyme molecular switches with blocking
The DNAzyme switches reported here comprise two partially complementary oligonucleotides; a DNAzyme capable of cleaving a nucleic acid substrate and a Blocking oligonucleotide (BL) (Scheme 1). The BL consists of two terminal inhibitory regions (A′ and B′), which are each complementary to a portion of the DNAzyme, and a third intermediate region, D, which does not hybridize to the DNAzyme. The intermediate region is universal and can, if desired, consist of any sequence that can be recognized and/or be modified by another molecule such as an enzyme. The BL can be designed so that it hybridizes with the DNAzyme in one of two different ways. Firstly, the 5′ and 3′ termini of the BL can be designed to hybridize with the 3′ and 5′ termini of the DNAzyme in a traditional manner to form a blocked duplexed DNAzyme structure (BL1). Alternatively, the 5′ and 3′ termini of the BL can be designed to hybridize with the 5′ and 3′ termini of the DNAzyme (BL2). In order to maintain correct Watson–Crick base pairing however, a quasi-circular structure is formed. In either conformation, the DNAzyme is initially temporarily inactive and unable to modify any substrates. In this study, an MNAzyme was utilized to recognize a target nucleic acid and activate the switches by cleaving the substrate within the intermediate region (Scheme 2). When this substrate is cleaved, it results in the truncation of the BL into two shorter fragments which can no longer hybridize efficiently to the DNAzyme under the same isothermal reaction conditions. Consequently, the DNAzyme is released and its catalytic activity is restored. The active DNAzyme can then cleave its nucleic acid substrate, which is dual-labelled with a fluorophore and quencher moiety, such that cleavage results in the production of a detectable fluorescent signal. For each of the two types of DNAzyme switches (Blocked duplexed DNAzymes or Quasi-circular DNAzymes), four different design variations (labelled Designs 1–4) were created. The detailed sequence designs are provided in Fig. S-1, Supplementary Data. For the blocked duplexed DNAzyme switches (Fig. S-1, panel A), Designs 1 and 2 contain all but three nucleotides of the DNAzyme sequence hybridized to the BL. These bases are located at approximately the center of the catalytic core. Designs 3 and 4 are similar to Designs 1 and 2, but instead contain five un-paired catalytic core nucleotides. Additionally, both Designs 2 and 4 contain two G-T wobble base pairs located approximately half-way within the center of each inhibitory BL region and the DNAzyme. Thus, each of the four designs are unique. The raw fluorescence data produced from reactions containing each design are shown in Fig. S-2 panel A, Supplementary Data. This data was normalized so that background signal (no target) was subtracted from the target signal and is presented in Fig. 1, panel A. The greatest signal-tobackground difference was produced from Design 2. This design provides an optimal balance with a higher melting temperature (Tm) at the center of the duplex to help keep the DNAzyme blocked in the absence of target, but with the inclusion of the additional G-T wobble base pairs to help reduce the total Tm slightly, increasing the ability of the two oligonucleotides to separate following cleavage of the BL by the MNAzyme. Design 4 did not have as great a signal to background difference as Design 2 because the combination of the G–T wobble base pairs with five un-paired bases lead to a greater instability between the two oligonucleotides under the assay conditions. Designs 1 and 3 on the other hand, were too stable under these conditions and therefore less favorable due to a lack of separation of the DNAzyme from the cleaved BL. For the quasi-circular DNAzyme switches (Fig. S-1, panel B), Designs 1–4 contain all but three, six, eight and twelve nucleotides of the DNAzyme sequence hybridized to the BL respectively. These
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Fig. 2. Quasi-circular DNAzyme switches can be utilized for molecular logic gates. (A) (i) An OR logic gate consisting of a quasi-circle comprising two adjacent substrates for Mz1 and Mz2. The presence of Target 1 or Target 2 results in the activation of Mz1 or Mz2 respectively. Cleavage of either Mz1 or Mz2 substrates by the corresponding MNAzyme results in DNAzyme activation from the switch as evidenced by the FAM output signal summarized in the truth table (ii) and shown in the raw data plot (iii). A comparable increase in fluorescence is observed with 1 nM of Targets 1 or 2, or 0.5 nM of each. (B) (i) A YES gate consists of a quasi-circular DNAzyme structure comprising two different BL (BLA and BLB) and two different DNAzymes (DzA and DzB), Cleavage of the MNAzyme substrate present within both BLA and BLB results in the simultaneous release and activation of both DzA and DzB. The active DNAzymes can then cleave their substrates which are fluorescently-labelled with FAM and Texas Red (TxR) fluorophores respectively. This results in an increase in both FAM and TxR output signals, as summarized in the truth table (ii) and shown in the raw data plot (iii).
un-paired bases are also located at approximately the center of the catalytic core. Additionally, Design 1 contains two G–T wobble base pairs located approximately half-way between the center of each inhibitory BL region and the DNAzyme. The raw fluorescence data produced from reactions with these designs is shown in Fig. S-2 panel B, Supplementary Data. The normalized signal is presented in Fig. 1, panel B. Here, the greatest signal to background difference was produced from Design 2 containing six un-paired catalytic core nucleotides. This was followed by Designs 1 and 3 which were not as favorable due to a higher level of background signal in each case. Design 4 with the lowest Tm, was highly nonspecific with very minimal difference between signal and background observed. The blocked duplexed and quasi-circular switches outlined here are unique for two reasons. Firstly, they temporarily inhibit a nucleic acid-modifying DNAzyme and secondly, in order to reactivate this DNAzyme they require the cleavage activity of another nucleic acid modifying enzyme (e.g. a DNAzyme or MNAzyme). Thus, the output of one switch can be used as the input of
another. This provides a significant advantage over existing methods in that they can be used to create circular feedback cascades (Section 3.3). In contrast, many existing methods utilize peroxidase-mimicking DNAzymes (Elbaz et al., 2009; Guo et al., 2010; Qiu et al., 2010; Zhu et al., 2011) which have not yet been demonstrated to catalyze modification of nucleic acid substrates. Additionally, other methods outlined to date may block nucleic acid-modifying DNAzymes with complementary oligonucleotides, however the DNAzyme is typically released the via a strand-displacing mechanism whereby the target simply hybridizes to the complementary (blocking) strand and does not cleave it (Deborggraeve et al., 2013; Kahan-Hanum et al., 2013; Orbach et al., 2012; Tian and Mao 2005; Wang et al., 2002). The two DNAzyme switches outlined here proved to be successful provided an optimal Tm existed between the two oligonucleotides for the reaction temperature. Theoretically, a higher reaction temperature would favor a stronger Tm and vice versa for a lower reaction temperature. This is of course within the limits of the enzymes temperature range for optimal catalytic activity. At the time of
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Fig. 3. Amplification of signal is achieved via the construction of cross-catalytic and auto-catalytic feedback cascades. (A) Circle A only. (B) A cross-catalytic cascade between Circles A and B. (C) An auto-catalytic cascade produced by Circle C. In each scenario, the MNAzyme (red) recognizes a target (green) and cleaves the Mz1 substrate (also red) which is present un-labelled within the intermediate region of each quasi-circular structure. Fluorescent signal is produced when Dz1 (blue) is released from a quasi-circle, where it then cleaves a fluorescently-labelled Dz1 substrate (also blue). The raw fluorescence data for each scenario is shown underneath the schematic, with signals shown from 100 pM of target (green), 20 pM of target (blue) and the no target control (black). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
development, the quasi-circular switches were more advanced and hence were used for further manipulations as described in Sections 3.2 and 3.3. 3.2. DNAzyme switches for molecular computational logic The ability to activate the DNAzyme switches in the presence of a specific target analyte and provide a detectable output also creates the potential for their use in molecular computation. Molecular computing elements hold great interest due to their potential to be exploited for logical control of biological processes (Orbach et al., 2012). Such systems may be used to function as “programmable drugs”, responding only in the presence of abnormal conditions (Elbaz et al., 2010a; Kahan-Hanum et al., 2013; Willner et al., 2012). To demonstrate potential for computation, we modified a quasi-circular DNAzyme switch (corresponding to Design 2, Fig. S-1, panel B) in one of two ways. In the first scenario (Fig. 2, panel A), we incorporated two adjacent substrate sequences within the intermediate region of the BL. Each substrate can be cleaved by a different MNAzyme recognizing a different target nucleic acid. MNAzyme 1 (Mz1) therefore recognizes Target 1 and cleaves the Mz1 substrate. Similarly, MNAzyme 2 (Mz2) recognizes Target 2 and cleaves the Mz2 substrate. An OR logic gate was constructed, whereby cleavage of at least one of the substrates present within the BL was all that was required for the release of the DNAzyme from the quasi-circle and the subsequent restoration of its catalytic activity. There was a comparable
increase in fluorescence signal when different targets were either individually provided or provided together at an equivalent concentration (the final concentration was 1 nM in each case). This indicates that cleavage at only one location within the intermediate region of the BL is all that is required for DNAzyme release. The inclusion of multiple adjacent substrates within the BL of the quasi-circular switch to create the OR logic gate is also advantageous for the creation of a circular feedback cascade (shown in Section 3.3). This is because the quasi-circle BL is designed to temporarily inactivate a DNAzyme and the addition of an initiator catalytic nucleic acid enzyme e.g. an MNAzyme that recognizes and cleaves the same substrate as the DNAzyme could be problematic since the initiator enzyme itself could compete with the DNAzyme for hybridization to the complementary regions of the BL. The two adjacent substrates therefore separate the MNAzyme initiation step from the subsequent DNAzyme catalysis and therefore minimize any unwanted binding. In the second scenario (Fig. 2, panel B), a unique quasi-circle was constructed containing two different DNAzyme molecules. In this case, a YES logic gate occurs with two possible outputs; i.e. both DNAzymes can be activated following cleavage of one of two BL molecules by an MNAzyme responsive to a single nucleic acid target (Target 1). The two different DNAzymes (DzA and DzB) were each hybridized to distinct terminal regions of the two BL molecules (BLA and BLB). Each BL contained the same MNAzyme substrate within its intermediate region. Cleavage of this substrate by
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the MNAzyme resulted in the release of DzA and DzB, which were capable of recognizing and cleaving the DzA and DzB substrates respectively. Here we chose to label these substrates with two different fluorophores; FAM for DzA substrate and Texas Red (TxR) for DzB substrate, so that the release of each DNAzyme from the quasi-circle was individually monitored by observing the fluorescence signal from each channel. When the target is present, an increase in fluorescence is observed above that of the no target control in both the FAM and TxR channels, indicating that both DzA and DzB are released from the switch. This therefore demonstrates that the quasi-circular switch can be scaled-up to incorporate multiple DNAzymes which can be activated by a single target. Such a switch may potentially increase the speed of a feedback cascade as the number of active DNAzymes would theoretically double with each activation event. 3.3. DNAzyme switches as components of signal amplification cascades In addition to logical operations, the DNAzyme switches developed herein can also be used as the basic components for signal amplification cascades. To demonstrate this, low picomolar concentrations of nucleic acid target were provided for an MNAzyme (Mz1), which can cleave an un-labelled substrate (Mz1 substrate) present within the intermediate region of the BL of a quasi-circular DNAzyme structure. Three different scenarios are presented in Fig. 3. In all three scenarios, a DNAzyme (Dz1) is hybridized to a BL. Fluorescent signal is produced when Dz1 is released from its BL and is then able to cleave a fluorescently-labelled Dz1 substrate. The first scenario (Fig. 3, panel A) consists of only a single quasicircle ‘Circle A’, which comprises a BL containing the substrate for Mz1 and an adjacent substrate for another DNAzyme (Dz2) which as such is not amenable to cleavage by activated DNAzyme Dz1. When Mz1 cleaves the Mz1 substrate, Dz1 is released and can only cleave the fluorescently-labelled Dz1 substrate, generating signal. Thus there is no potential for additional DNAzymes to be activated. Consequently, in this scenario, there was only a slow and linear increase in fluorescence signal with 100 pM target and no detectable increase in signal for 20 pM of target above the no target control. In the second scenario (Fig. 3, panel B), a cross-catalytic cascade between Circle A and a different quasi-circle, ‘Circle B’ was constructed as a means to promote amplification of signal. In this instance, Dz1 when released from Circle A, also has the potential to cleave an un-labelled Dz1 substrate present within the intermediate region of the BL of Circle B and vice versa for the DNAzyme (Dz2) of Circle B. Each time this occurs, additional Dz1 and Dz2 molecules are released and their catalytic activity restored. In this scenario, the signal for 100 pM of target increased at a faster rate and reached a plateau much sooner than the corresponding non-cascade reaction in Fig. 3 panel A. The 20 pM target signal was also now detectable above the no target control. Alternatively, the third scenario involved the construction of an auto-catalytic cascade containing only one quasi-circle ‘Circle C’ (Fig. 3 panel C)). In this instance, the DNAzyme that was initially hybridized to the terminal regions of the BL (Dz1), also had the potential to bind and cleave a substrate within the intermediate region of the same BL molecule. The auto-catalytic quasi-circle also contained the substrate for Mz1, thus initiating the cascade in a target-dependent manner. The auto-catalytic cascade provided by Circle C also resulted in fluorescent signals for 100 pM and 20 pM target concentrations which reached a plateau much sooner than that of the corresponding non-cascade reaction in Fig. 3 panel A. These signals were both also faster than the cross-catalytic cascade shown in Fig. 3 panel B. The auto-catalytic ability of the quasicircle switch was primarily achieved by its unique design in that
there is greater complementarity between the DNAzyme and the 5′ and 3′ ends of the BL (regions A′ and B′ from Scheme 1) rather than the internal substrate sequence with which it also shares complementarity (region D from Scheme 1). This therefore means that in addition to the substrate-binding arms, part of the DNAzyme core sequence is also designed to hybridize with the noncleaved BL. The advantage to creating an auto-catalytic system over that of a cross-catalytic system was that the reaction components were effectively halved and there was no unwanted complementarity between different DNAzymes and BL molecules such as that which could occur in a cross-catalytic cascade. This allowed us to use lower BL to DNAzyme ratios, making reactions proceed faster. We have therefore demonstrated that the DNAzyme switches outlined here can be successfully incorporated into circular feedback cascades for the purpose of signal amplification. This is primarily due to their unique design requiring a nucleic acid-cleaving input and producing a nucleic acid-cleaving DNAzyme as an output. Thus, each individual switch possesses the ability to feed into one another creating a molecular ‘domino effect’. Previous switching attempts have largely utilized peroxidase-mimicking DNAzymes as the output, however, despite the broad applications of peroxidase-mimicking DNAzymes, these enzymes cannot catalyze the modification of nucleic acid substrates, thus they provide no mechanism for activation of additional catalytic nucleic acids capable of mediating signal amplification. The cascades produced here have advantages due to their ability to be responsive to different types of analytes (e.g. nucleic acids, small molecules, metal ions) (Bone et al., 2014). Additional advantages of the approach including the capacity for single nucleotide discrimination and multiplexing were demonstrated by our recent use of the autocatalytic quasi-circle switch in a ‘DNA-only cascade’ (Bone et al., 2014). From that study, the detection limit for nucleic acid targets was calculated to be 2.3 pM, which is comparable to other DNAzyme-based and protein enzyme-free methods reported, but provides a significant improvement in detection speed as many of these methods require much longer incubation times of up to 12 h (Bone et al., 2014). In addition, our method can be performed within a single-tube, with a single-step protocol. Future improvements to sensitivity are hypothesized to involve further optimization of the switch design and the oligonucleotide concentrations.
4. Conclusion This paper outlines the construction of nucleic acid-modifying DNAzyme switches. The DNAzymes were temporarily inactivated via Watson–Crick hybridization to complementary regions of blocking oligonucleotides and restoration of DNAzyme catalytic activity was then achieved via modification of an intermediate region between the two DNAzyme complementary regions of the BL, which only occurred in the presence of a specific target analyte. The switches can be easily modified for the purposes of both molecular computation and for constructing circular feedback cascades in order to achieve signal amplification, conferring significant potential for biosensing applications.
Acknowledgements This work was supported by SpeeDx Pty Ltd. The authors would like to acknowledge Dr. Elisa Mokany and Nicole Hasick for their technical assistance.
S.M. Bone et al. / Biosensors and Bioelectronics 70 (2015) 330–337
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.03.057.
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