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A dual input DNA-based molecular switch† Cite this: Mol. BioSyst., 2014, 10, 2810
Irina V. Nesterova, Siddieg O. Elsiddieg and Evgueni E. Nesterov
Received 19th June 2014, Accepted 22nd July 2014 DOI: 10.1039/c4mb00363b www.rsc.org/molecularbiosystems
We have designed and characterized a DNA-based molecular switch which processes two physiologically relevant inputs: pH (i.e. alkalinisation) and enzymatic activity, and generates a chemical output (in situ synthesized oligonucleotide). The design, based on allosteric interactions between i-motif and hairpin stem within the DNA molecule, addresses such critical physiological system parameters as molecular simplicity, tunability, orthogonality of the two input sensing domains, and compatibility with intracellular operation/delivery.
Better understanding and control of complex physiological processes requires availability of biocompatible tools which can be specifically tuned to examine relevant conditions. DNA-based molecular devices capable to operate inside living systems represent a promising platform for the development of such tools.1–3 DNA is a naturally biocompatible molecule with recognition capacity for a variety of targets (ranging from ions to small molecules to oligonucleotides and proteins/peptides to whole cells), and established approaches for controlling binding affinities, selectivity, stability, etc. An attractive feature of DNA-based systems is programmability which allows designing and implementing molecular level logic circuitry.4 Indeed, a number of DNA-based programmable nanosystems performing molecular computing functions inside living systems have been demonstrated.5 Moreover, recent advancements in nucleic acids delivery technologies6 provide strong support for the viability of this approach and boost further developments in the area. However, some significant shortcomings still remain to be addressed. The major one is a limited variety of inputs used so far as the previously reported devices were mostly based on complex RNA-related inputs and/or specific aptamerdependent stimuli. At the same time, it has been shown that
Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, USA. E-mail: [email protected] † Electronic supplementary information (ESI) available: Experimental procedures. See DOI: 10.1039/c4mb00363b
2810 | Mol. BioSyst., 2014, 10, 2810--2814
RNA-expression signatures in many cases are not the only reliable indicators–predictors of complex intracellular conditions;7 and control over variety of other endogenous traits circumventing redundancies in RNA/DNA expression levels should be explored. Those traits may include environmental signatures, ion concentration, enzymatic activities, etc. Another persisting problem is complexity and large size of typical DNA-based devices. Incorporation of multiple input/output functional modules tends to increase bulkiness of the resulting molecular systems. The excessive complexity, in turn, jeopardizes the practical utility of the agents amid concerns over reliability of their intracellular assembly as well as delivery of large DNA constructs across cellular membranes.8,9 Recently we have developed an allosteric control mechanism involving cooperative interactions between two functional domains: a quadruplex and a hairpin stem.10 This mechanism makes possible to incorporate two orthogonal sensing modules within a simple single-stranded DNA molecular construct. Importantly, the allosteric mechanism provides both thermodynamic and kinetic tools to manipulate operational parameters of both domains. In this Communication, we report on the design and evaluation of a DNA molecular switch based on cooperative interactions between an i-motif and a hairpin stem. i-Motif is a tetraplex structure mediated by the hemiprotonated cytidine–H+–cytidine base pairs.11 An i-motif can be formed by single stranded oligonucleotides containing at least four tracts of cytosines; and its conformational stability is pH-dependent.12 The i-motif-based isothermally operating switch described herein processes two physiologically relevant orthogonal inputs: pH (environment alkalinisation) as an input 1 and polymerase activity as an input 2, and generates a chemical output (double-stranded DNA) only when both inputs are present. The molecular device (evaluated in vitro) was specifically designed to undergo transition over a physiologically relevant range (i.e. transition pH 7.2 in the proof-of-concept model) and shows potential for tunability. We also emphasize the molecular simplicity of the construct achieved via using a short single stranded intramolecular folded structure to maximize compatibility with intracellular operation/delivery.9
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Fig. 1 Schematic of the dual input allosteric molecular switch Ii. The single stranded DNA Ii contains a pH-sensitive module (i-motif, domain 2), a primer for downstream polymerase extension (domain 1a–1), a spacer (domain 3), and a protective sequence (domain 1*). When pH (input 1) is below the target value of 7.2 (pH o 7.2, right pathway), folding of the i-motif in Ii stabilizes a hairpin stem. As a result, primer domain 1a–1 is prevented from hybridizing template II therefore producing no extension product III (output) in the presence of polymerase (input 2). In contrast, when pH (input 1) is above the target value of 7.2 (pH 4 7.2, left pathway), the i-motif unfolding destabilizes the stem in hairpin Ii. As a result, Ii forms a random coil which makes the primer domain 1a–1 available for hybridizing a complementary domain 1*–1a* of the template II. In such a case, and only when polymerase (input 2) is present, the system yields the double stranded extension product III (output). The arrow on DNA pictograms indicates 3 0 direction.
The proof-of-concept single-stranded device Ii consists of the following functional modules (Fig. 1):13 an 11 bp primer for a downstream polymerase extension (domain 1a–1); a 6 bp stem (domain 1*) complementary to the sub-domain 1 of the primer and designed to protect the primer from hybridizing complementary template when hairpin stem is closed; a spacer consisting of 4 adenines (domain 3) to position the folded fragment in the middle of the loop; and an i-motif fragment (domain 2). In the model system, the structure of the i-motif fragment was specifically adjusted for it to undergo folding– unfolding transition at pH 7.2 (Fig. 2). Unfolding the i-motif at pH 4 7.2 (input 1 present, left pathway in Fig. 1) forces the device into a random coil. This makes the primer (domain 1a–1) available for hybridizing a complementary region (domain 1*–1a*) of a DNA template II. In such a case, and only with the sufficient level of polymerase activity (input 2) present, the system will synthesize a double extension product III as an output (in the presence of dNTPs). When the i-motif is folded (pH o 7.2, input 1 absent, right pathway in Fig. 1), Ii exists as a
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Molecular BioSystems
Fig. 2 pH-dependent configuration changes of oligonucleotide Ii are detected via CD (a) and UV (b). (c) CD signal at 290 nm (black squares) and UV signal at 295 nm (grey circles) of Ii as a function of pH. Experimental details are included in ESI.†
stable hairpin structure because folding of the intraloop i-motif allosterically stabilizes the hairpin over the corresponding random coil conformation therefore effectively preventing the primer (domain 1a–1) from hybridizing the template II and precluding the enzymatic extension (even if the input 2 – polymerase – is present). Obviously, in the absence of input 2 (polymerase activity) no output will be generated independent on the status of input 1 (value of pH). Overall, the whole system behaves as a two-input-one-output molecular device with two independent inputs (increased pH and polymerase activity) required to be present simultaneously to generate an ultimate output – a double-stranded DNA product. We evaluated in vitro performance of the device Ii in response to the action of two inputs: pH (above and below the pre-set value of 7.2) and polymerase activity.14 To impose an appropriate pH input, we carried out the studies in two buffer solutions: New England Biolabs’ Buffer 1 (NEB 1) and Buffer 2 (NEB 2) since both buffers have physiologically relevant composition deemed appropriate for polymerase extensions, and show pH values flanking the targeted transition range (pH 7.0 for NEB 1 and pH 7.9 for NEB 2). In principle, concentrations of the device Ii and the template II may affect propensity of the primer:template duplex formation. We deliberately eliminated
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this variable by keeping concentrations of Ii and II constant throughout the experiments. To conclusively address any effects other than intended Ii’s structural transformations on output generation, we carefully designed and evaluated several controls: (1) an 11 bp primer p (1a–1) identical to the Ii’s primer domain; (2) a hairpin Ih (1*–3–1a–1), with a hairpin stem identical to one in Ii and a short loop representing an ‘‘apparent’’ loop of device Ii when i-motif is folded; and (3) a hairpin Is (1*–3–2a–1a–1), with a stem identical to both Ii and Ih and a full-length loop of the size identical to device Ii but without i-motif forming sequence to account for any potential effects from long unstructured loop of the device.13 The design considerations included two major requirements: (1) the duplex between primer and template should be stable enough to initiate polymerase extension reaction when primer is available for target hybridization – as predicted by mfold, the Tm of the duplex used in Ii and related controls was 40 1C; (2) the Ii structure should be stable in acidic environments to discriminate between two pH conditions. To estimate the Ii’s stability we evaluated its Tm in NEB 1 and NEB 2. Since pKa of NEB 1 and NEB 2 depends on temperature,15 we did not conduct any detailed thermodynamic analysis based on these experiments other than simple estimating Ii’s Tm in the actual experimental conditions. The Tm of Ii in NEB 1 was B45 1C while no melting transition was observed in NEB 2. As a model for the enzymatic activity input (input 2), we chose an isothermally operating Bst polymerase. We monitored formation of the double-stranded product III (output) via an enhancement of SYBR Green I fluorescence (Fig. 3a–d) as well as using non-denaturing polyacrylamide gel electrophoresis (PAGE) (Fig. 3e–h). First, we evaluated differences in intrinsic activity of Bst polymerase in NEB 1 and NEB 2. According to the manufacturer, the Bst large fragment polymerase is more active in NEB 2.16 This trend indeed was observed to some (however rather insignificant) extent in fluorescent studies: slightly higher emission in NEB 2 was detected from the extension product of control oligonucleotides p (Fig. 3a) and Is (Fig. 3c). However, in PAGE experiments we observed no difference between the two buffers
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for controls p and Is (Fig. 3e and g, respectively). Therefore, we can safely assume that in our experiments the effect of buffer on polymerase extension was insignificant and all the experimental observations reported herein originated from appropriate structural transformations of the evaluated oligonucleotides. In excellent agreement with our model, device Ii demonstrated input-specific generation of an output extension product: the extension product III was clearly detected in NEB 2 (pH 7.9, i.e. input 1 present) with added polymerase (input 2 present) by both fluorescence and PAGE whereas no extension product III was detected in NEB 1 (pH 7.0, i.e. input 1 absent) independent on the presence or absence of the input 2 (Fig. 3d and h, respectively). Similarly, no output extension product was detected from the device in NEB 2 (input 1 present) in the absence of polymerase (input 2 absent). Therefore, the device Ii unambiguously demonstrated dual-input specific switching capabilities under evaluated conditions. Controls further confirmed that the output was produced exclusively owing to conformational transitions of Ii. Thus, primer p (a simple primer with no responsive built-in capabilities) produced an extension product III in both buffers (Fig. 3a and e) demonstrating (as discussed above) no significant effect from the system components on polymerase extension. No output product III in both buffers was detected for a short unstructured hairpin Ih indicating efficient protection of the primer domain in the closed hairpin (Fig. 3b and f). In contrast, the long-loop control device Is indiscriminately produced the extension product III in both buffers (Fig. 3c and g) in a clear demonstration of the lack of primer protection in the large unstructured hairpin. Overall, we demonstrated that the allosteric control mechanism based on cooperative interactions between a hairpin and an i-motif can be utilized in the design of efficiently operating molecular switches. Importantly, the allosterically controlled system provides: (i) molecular simplicity, (ii) an opportunity to combine two orthogonal sensing modalities within a simple single-stranded system, and (iii) a set of thermodynamic and kinetic tools to manipulate the operation parameters of those modalities. The tools may, for example, be used to achieve
Fig. 3 Detection of double stranded extension product III in the presence of device Ii (d, h) and controls p (a, e), Ih (b, f) and Is (c, g) via fluorescence of SYBR Green (F in a.u., a–d), and PAGE after staining with SYBR Gold (e–h). Electrophoresis lines assignments: L – 50 bp double stranded DNA ladder with corresponding band identities; 1, 5, 9 and 13 – NEB 1 and no Bst polymerase; 2, 6, 10, and 14 – NEB 2 and no Bst polymerase; 3, 7, 11, 15 – NEB 1 and Bst polymerase present; 4, 8, 12 and 16 – NEB 2 and Bst polymerase present. Horizontal line in graphs a–d indicates suggested threshold value for fluorescence. Experimental details are included in ESI.†
2812 | Mol. BioSyst., 2014, 10, 2810--2814
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precise tuning of pH switching range17 and/or manipulate a primer protection strength, a capability inaccessible with more traditional approaches utilized in input-triggered devices based on regular short hairpins.18 These advantages originate from a more favorable DG1 values for stems of quadruplex-containing hairpins (at temperatures below Tm) compared to a short hairpin without inserted quadruplex with identical stem and loop length (as was demonstrated by us earlier).10 Overall, implementation of the allosteric mechanism significantly expands the arsenal of tools for rational design of DNA-based molecular devices with defined operational parameters. Although the presented system is an in vitro model, this approach (with appropriate adjustments for molecular crowding19) may eventually become extrapolated toward intracellular applications with the involvement of biochemically or clinically relevant enzymes (e.g. telomerase). The molecular simplicity and compact nature of the presented device should help circumvent concerns over its intracellular delivery. General compatibility of i-motif systems with nuclease resistant DNA backbone modification should in principle address concerns over the device stability in biological media.20 Programmability (a characteristic of DNA-based scaffolds) enables device adjustment for particular targeted conditions. Therefore, considering that i-motif based sensors have already been demonstrated to function inside cells2 and living systems,3 the present findings demonstrate potential of this approach to develop powerful tools for assessment of different conditions in biological environments.
Conclusions In conclusion, we have designed and evaluated a simple allosteric single-stranded DNA-based dual-input switch operating in physiologically relevant conditions. The switch utilizes intracellularlyrelevant triggers such as small change in pH and presence of polymerase activity as two inputs and synthesizes in situ a chemical output (oligonucleotide fragment) only when both inputs are present. It can be a viable alternative to molecular devices that rely on mRNA expressions or other indicators associated with redundant pathways, as well as devices which have an output moiety already incorporated as part of their molecular structure. We anticipate that the device architecture based on manipulating hairpin stability via inserted quadruplexed structures can be extended to confer other appropriate triggers.
Acknowledgements This work was supported by LSU College of Science. Purchase of CD spectrometer was funded by the National Science Foundation CRIF grant CHE-0840516.
Notes and references 1 C. Battle, X. Chu and J. Jayawickramarajah, Supramol. Chem., 2013, 25, 848; T. S. Bayer and C. D. Smolke, Nat. Biotechnol., 2005, 23, 337; A. R. Buskirk, A. Landrigan and D. R. Liu, Chem. Biol., 2004, 11, 1157.
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2 S. Modi, C. Nizak, S. Surana, S. Halder and Y. Krishnan, Nat. Nanotechnol., 2013, 8, 459; S. Modi, M. G. Swetha, D. Goswami, G. D. Gupta, S. Mayor and Y. Krishnan, Nat. Nanotechnol., 2009, 4, 325. 3 S. Surana, J. M. Bhat, S. P. Koushika and Y. Krishnan, Nat. Commun., 2011, 2, 7. 4 S. M. Douglas, I. Bachelet and G. M. Church, Science, 2012, 335, 831; Z. Z. Huang, J. S. Ren and X. G. Qu, Mol. BioSyst., 2012, 8, 921; J. Guo and R. Q. Yang, Mol. BioSyst., 2012, 8, 927. 5 F. Wang, C. H. Lu and I. Willner, Chem. Rev., 2014, 114, 2881; H. Pei, L. Liang, G. Yao, J. Li, Q. Huang and C. Fan, Angew. Chem., Int. Ed., 2012, 51, 9020; M. Leisner, L. Bleris, J. Lohmueller, X. Zhen and Y. Benenson, Nat. Nanotechnol., 2010, 5, 666; K. Rinaudo, L. Bleris, R. Maddamsetti, S. Subramanian, R. Weiss and Y. Benenson, Nat. Biotechnol., 2007, 25, 795; E. Shapiro and B. Gil, Science, 2008, 322, 387; Z. Xie, L. Wroblewska, L. Prochazka, R. Weiss and Y. Benenson, Science, 2011, 333, 1307; C. J. Delebecque, A. B. Lindner, P. A. Silver and F. A. Aldaye, Science, 2011, 333, 470; B. Gil, M. KahanHanum, N. Skirtenko, R. Adar and E. Shapiro, Nano Lett., 2011, 11, 2989; M. N. Win and C. D. Smolke, Science, 2008, 322, 456. 6 K. Watanabe, M. Harada-Shiba, A. Suzuki, R. Gokuden, R. Kurihara, Y. Sugao, T. Mori, Y. Katayama and T. Niidome, Mol. BioSyst., 2009, 5, 1306; M. Mahato, P. Kumar and A. K. Sharma, Mol. BioSyst., 2013, 9, 780; Acc. Chem. Res., Special Issue on Gene Silencing and Delivery, 2012, 45(7), 959–1172; L. Cui, J. L. Cohen, C. K. Chu, ´chet, J. Am. Chem. P. R. Wich, P. H. Kierstead and J. M. J. Fre Soc., 2012, 134, 15840; V. Sokolova and M. Epple, Angew. Chem., Int. Ed., 2008, 47, 1382; R. L. Juliano, X. Ming and O. Nakagawa, Bioconjugate Chem., 2012, 23, 147. 7 W. S. Dalton and S. H. Friend, Science, 2006, 312, 1165; D. Hanahan and R. A. Weinberg, Cell, 2011, 144, 646; E. E. Schadt, Nature, 2009, 461, 218. 8 A. V. Pinheiro, D. Han, W. M. Shih and H. Yan, Nat. Nanotechnol., 2011, 6, 763. 9 F. C. Simmel, Nanomedicine, 2007, 2, 817. 10 I. V. Nesterova, S. O. Elsiddieg and E. E. Nesterov, J. Phys. Chem. B, 2013, 117, 10115. 11 S. Ahmed, A. Kintanar and E. Henderson, Nat. Struct. Biol., 1994, 1, 83; K. Gehring, J. L. Leroy and M. Gueron, Nature, ´ron, J. L. Mergny and 1993, 363, 561; J. L. Leroy, M. Gue ´le `ne, Nucleic Acids Res., 1994, 22, 1600. C. He ´le `ne, 12 J. L. Mergny, L. Lacroix, X. G. Han, J. L. Leroy and C. He J. Am. Chem. Soc., 1995, 117, 8887. 13 Complete sequences of Ii, Ih, Is, p, i, and Target II are included in Table S1 in ESI†. 14 Details of experimental design and procedures are included in ESI†. 15 A. A. El-Harakany, F. M. Abdel Halim and A. O. Barakat, J. Electroanal. Chem. Interfacial Electrochem., 1984, 162, 285. 16 Available from: http://www.neb.com/nebecomm/products/ faqproductM0275.asp#180.
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17 I. V. Nesterova and E. E. Nesterov, J. Am. Chem. Soc., 2014, 136, 8843. 18 Y. Weizmann, M. K. Beissenhirtz, Z. Cheglakov, R. Nowarski, M. Kotler and I. Willner, Angew. Chem., Int. Ed., 2006, 45, 7384; A. R. Connolly and M. Trau, Angew. Chem., Int. Ed., 2010, 49, 2720. 19 A. Rajendran, S.-i. Nakano and N. Sugimoto, Chem. Commun., 2010, 46, 1299. 20 N. Kumar, M. Petersen and S. Maiti, Chem. Commun., 2009, 1532; C. C. Hardin, M. Corregan, B. A. Brown and L. N. Frederick, Biochemistry, 1993, 32, 5870; J. L. Mergny
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and L. Lacroix, Nucleic Acids Res., 1998, 26, 4797; H. Kanehara, M. Mizuguchi, K. Tajima, K. Kanaori and K. Makino, Biochemistry, 1997, 36, 1790; N. K. Sharma and K. N. Ganesh, Chem. Commun., 2005, 4330; Y. Krishnan-Ghosh, E. Stephens and S. Balasubramanian, Chem. Commun., 2005, 5278; J. A. Brazier, J. Fisher and R. Cosstick, Angew. Chem., Int. Ed., 2006, 45, 114; N. Kumar, J. T. Nielsen, S. Maiti and M. Petersen, Angew. Chem., Int. Ed., 2007, 46, 9220; C. P. Fenna, V. J. Wilkinson, J. R. P. Arnold, R. Cosstick and J. Fisher, Chem. Commun., 2008, 3567; Y. Zhao, L. Cao, J. Ouyang, M. Wang, K. Wang and X. H. Xia, Anal. Chem., 2013, 85, 1053.
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A dual input DNA-based molecular switch† Cite this: Mol. BioSyst., 2014, 10, 2810
Irina V. Nesterova, Siddieg O. Elsiddieg and Evgueni E. Nesterov
Received 19th June 2014, Accepted 22nd July 2014 DOI: 10.1039/c4mb00363b www.rsc.org/molecularbiosystems
We have designed and characterized a DNA-based molecular switch which processes two physiologically relevant inputs: pH (i.e. alkalinisation) and enzymatic activity, and generates a chemical output (in situ synthesized oligonucleotide). The design, based on allosteric interactions between i-motif and hairpin stem within the DNA molecule, addresses such critical physiological system parameters as molecular simplicity, tunability, orthogonality of the two input sensing domains, and compatibility with intracellular operation/delivery.
Better understanding and control of complex physiological processes requires availability of biocompatible tools which can be specifically tuned to examine relevant conditions. DNA-based molecular devices capable to operate inside living systems represent a promising platform for the development of such tools.1–3 DNA is a naturally biocompatible molecule with recognition capacity for a variety of targets (ranging from ions to small molecules to oligonucleotides and proteins/peptides to whole cells), and established approaches for controlling binding affinities, selectivity, stability, etc. An attractive feature of DNA-based systems is programmability which allows designing and implementing molecular level logic circuitry.4 Indeed, a number of DNA-based programmable nanosystems performing molecular computing functions inside living systems have been demonstrated.5 Moreover, recent advancements in nucleic acids delivery technologies6 provide strong support for the viability of this approach and boost further developments in the area. However, some significant shortcomings still remain to be addressed. The major one is a limited variety of inputs used so far as the previously reported devices were mostly based on complex RNA-related inputs and/or specific aptamerdependent stimuli. At the same time, it has been shown that
Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, USA. E-mail: [email protected] † Electronic supplementary information (ESI) available: Experimental procedures. See DOI: 10.1039/c4mb00363b
2810 | Mol. BioSyst., 2014, 10, 2810--2814
RNA-expression signatures in many cases are not the only reliable indicators–predictors of complex intracellular conditions;7 and control over variety of other endogenous traits circumventing redundancies in RNA/DNA expression levels should be explored. Those traits may include environmental signatures, ion concentration, enzymatic activities, etc. Another persisting problem is complexity and large size of typical DNA-based devices. Incorporation of multiple input/output functional modules tends to increase bulkiness of the resulting molecular systems. The excessive complexity, in turn, jeopardizes the practical utility of the agents amid concerns over reliability of their intracellular assembly as well as delivery of large DNA constructs across cellular membranes.8,9 Recently we have developed an allosteric control mechanism involving cooperative interactions between two functional domains: a quadruplex and a hairpin stem.10 This mechanism makes possible to incorporate two orthogonal sensing modules within a simple single-stranded DNA molecular construct. Importantly, the allosteric mechanism provides both thermodynamic and kinetic tools to manipulate operational parameters of both domains. In this Communication, we report on the design and evaluation of a DNA molecular switch based on cooperative interactions between an i-motif and a hairpin stem. i-Motif is a tetraplex structure mediated by the hemiprotonated cytidine–H+–cytidine base pairs.11 An i-motif can be formed by single stranded oligonucleotides containing at least four tracts of cytosines; and its conformational stability is pH-dependent.12 The i-motif-based isothermally operating switch described herein processes two physiologically relevant orthogonal inputs: pH (environment alkalinisation) as an input 1 and polymerase activity as an input 2, and generates a chemical output (double-stranded DNA) only when both inputs are present. The molecular device (evaluated in vitro) was specifically designed to undergo transition over a physiologically relevant range (i.e. transition pH 7.2 in the proof-of-concept model) and shows potential for tunability. We also emphasize the molecular simplicity of the construct achieved via using a short single stranded intramolecular folded structure to maximize compatibility with intracellular operation/delivery.9
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Fig. 1 Schematic of the dual input allosteric molecular switch Ii. The single stranded DNA Ii contains a pH-sensitive module (i-motif, domain 2), a primer for downstream polymerase extension (domain 1a–1), a spacer (domain 3), and a protective sequence (domain 1*). When pH (input 1) is below the target value of 7.2 (pH o 7.2, right pathway), folding of the i-motif in Ii stabilizes a hairpin stem. As a result, primer domain 1a–1 is prevented from hybridizing template II therefore producing no extension product III (output) in the presence of polymerase (input 2). In contrast, when pH (input 1) is above the target value of 7.2 (pH 4 7.2, left pathway), the i-motif unfolding destabilizes the stem in hairpin Ii. As a result, Ii forms a random coil which makes the primer domain 1a–1 available for hybridizing a complementary domain 1*–1a* of the template II. In such a case, and only when polymerase (input 2) is present, the system yields the double stranded extension product III (output). The arrow on DNA pictograms indicates 3 0 direction.
The proof-of-concept single-stranded device Ii consists of the following functional modules (Fig. 1):13 an 11 bp primer for a downstream polymerase extension (domain 1a–1); a 6 bp stem (domain 1*) complementary to the sub-domain 1 of the primer and designed to protect the primer from hybridizing complementary template when hairpin stem is closed; a spacer consisting of 4 adenines (domain 3) to position the folded fragment in the middle of the loop; and an i-motif fragment (domain 2). In the model system, the structure of the i-motif fragment was specifically adjusted for it to undergo folding– unfolding transition at pH 7.2 (Fig. 2). Unfolding the i-motif at pH 4 7.2 (input 1 present, left pathway in Fig. 1) forces the device into a random coil. This makes the primer (domain 1a–1) available for hybridizing a complementary region (domain 1*–1a*) of a DNA template II. In such a case, and only with the sufficient level of polymerase activity (input 2) present, the system will synthesize a double extension product III as an output (in the presence of dNTPs). When the i-motif is folded (pH o 7.2, input 1 absent, right pathway in Fig. 1), Ii exists as a
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Molecular BioSystems
Fig. 2 pH-dependent configuration changes of oligonucleotide Ii are detected via CD (a) and UV (b). (c) CD signal at 290 nm (black squares) and UV signal at 295 nm (grey circles) of Ii as a function of pH. Experimental details are included in ESI.†
stable hairpin structure because folding of the intraloop i-motif allosterically stabilizes the hairpin over the corresponding random coil conformation therefore effectively preventing the primer (domain 1a–1) from hybridizing the template II and precluding the enzymatic extension (even if the input 2 – polymerase – is present). Obviously, in the absence of input 2 (polymerase activity) no output will be generated independent on the status of input 1 (value of pH). Overall, the whole system behaves as a two-input-one-output molecular device with two independent inputs (increased pH and polymerase activity) required to be present simultaneously to generate an ultimate output – a double-stranded DNA product. We evaluated in vitro performance of the device Ii in response to the action of two inputs: pH (above and below the pre-set value of 7.2) and polymerase activity.14 To impose an appropriate pH input, we carried out the studies in two buffer solutions: New England Biolabs’ Buffer 1 (NEB 1) and Buffer 2 (NEB 2) since both buffers have physiologically relevant composition deemed appropriate for polymerase extensions, and show pH values flanking the targeted transition range (pH 7.0 for NEB 1 and pH 7.9 for NEB 2). In principle, concentrations of the device Ii and the template II may affect propensity of the primer:template duplex formation. We deliberately eliminated
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this variable by keeping concentrations of Ii and II constant throughout the experiments. To conclusively address any effects other than intended Ii’s structural transformations on output generation, we carefully designed and evaluated several controls: (1) an 11 bp primer p (1a–1) identical to the Ii’s primer domain; (2) a hairpin Ih (1*–3–1a–1), with a hairpin stem identical to one in Ii and a short loop representing an ‘‘apparent’’ loop of device Ii when i-motif is folded; and (3) a hairpin Is (1*–3–2a–1a–1), with a stem identical to both Ii and Ih and a full-length loop of the size identical to device Ii but without i-motif forming sequence to account for any potential effects from long unstructured loop of the device.13 The design considerations included two major requirements: (1) the duplex between primer and template should be stable enough to initiate polymerase extension reaction when primer is available for target hybridization – as predicted by mfold, the Tm of the duplex used in Ii and related controls was 40 1C; (2) the Ii structure should be stable in acidic environments to discriminate between two pH conditions. To estimate the Ii’s stability we evaluated its Tm in NEB 1 and NEB 2. Since pKa of NEB 1 and NEB 2 depends on temperature,15 we did not conduct any detailed thermodynamic analysis based on these experiments other than simple estimating Ii’s Tm in the actual experimental conditions. The Tm of Ii in NEB 1 was B45 1C while no melting transition was observed in NEB 2. As a model for the enzymatic activity input (input 2), we chose an isothermally operating Bst polymerase. We monitored formation of the double-stranded product III (output) via an enhancement of SYBR Green I fluorescence (Fig. 3a–d) as well as using non-denaturing polyacrylamide gel electrophoresis (PAGE) (Fig. 3e–h). First, we evaluated differences in intrinsic activity of Bst polymerase in NEB 1 and NEB 2. According to the manufacturer, the Bst large fragment polymerase is more active in NEB 2.16 This trend indeed was observed to some (however rather insignificant) extent in fluorescent studies: slightly higher emission in NEB 2 was detected from the extension product of control oligonucleotides p (Fig. 3a) and Is (Fig. 3c). However, in PAGE experiments we observed no difference between the two buffers
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for controls p and Is (Fig. 3e and g, respectively). Therefore, we can safely assume that in our experiments the effect of buffer on polymerase extension was insignificant and all the experimental observations reported herein originated from appropriate structural transformations of the evaluated oligonucleotides. In excellent agreement with our model, device Ii demonstrated input-specific generation of an output extension product: the extension product III was clearly detected in NEB 2 (pH 7.9, i.e. input 1 present) with added polymerase (input 2 present) by both fluorescence and PAGE whereas no extension product III was detected in NEB 1 (pH 7.0, i.e. input 1 absent) independent on the presence or absence of the input 2 (Fig. 3d and h, respectively). Similarly, no output extension product was detected from the device in NEB 2 (input 1 present) in the absence of polymerase (input 2 absent). Therefore, the device Ii unambiguously demonstrated dual-input specific switching capabilities under evaluated conditions. Controls further confirmed that the output was produced exclusively owing to conformational transitions of Ii. Thus, primer p (a simple primer with no responsive built-in capabilities) produced an extension product III in both buffers (Fig. 3a and e) demonstrating (as discussed above) no significant effect from the system components on polymerase extension. No output product III in both buffers was detected for a short unstructured hairpin Ih indicating efficient protection of the primer domain in the closed hairpin (Fig. 3b and f). In contrast, the long-loop control device Is indiscriminately produced the extension product III in both buffers (Fig. 3c and g) in a clear demonstration of the lack of primer protection in the large unstructured hairpin. Overall, we demonstrated that the allosteric control mechanism based on cooperative interactions between a hairpin and an i-motif can be utilized in the design of efficiently operating molecular switches. Importantly, the allosterically controlled system provides: (i) molecular simplicity, (ii) an opportunity to combine two orthogonal sensing modalities within a simple single-stranded system, and (iii) a set of thermodynamic and kinetic tools to manipulate the operation parameters of those modalities. The tools may, for example, be used to achieve
Fig. 3 Detection of double stranded extension product III in the presence of device Ii (d, h) and controls p (a, e), Ih (b, f) and Is (c, g) via fluorescence of SYBR Green (F in a.u., a–d), and PAGE after staining with SYBR Gold (e–h). Electrophoresis lines assignments: L – 50 bp double stranded DNA ladder with corresponding band identities; 1, 5, 9 and 13 – NEB 1 and no Bst polymerase; 2, 6, 10, and 14 – NEB 2 and no Bst polymerase; 3, 7, 11, 15 – NEB 1 and Bst polymerase present; 4, 8, 12 and 16 – NEB 2 and Bst polymerase present. Horizontal line in graphs a–d indicates suggested threshold value for fluorescence. Experimental details are included in ESI.†
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precise tuning of pH switching range17 and/or manipulate a primer protection strength, a capability inaccessible with more traditional approaches utilized in input-triggered devices based on regular short hairpins.18 These advantages originate from a more favorable DG1 values for stems of quadruplex-containing hairpins (at temperatures below Tm) compared to a short hairpin without inserted quadruplex with identical stem and loop length (as was demonstrated by us earlier).10 Overall, implementation of the allosteric mechanism significantly expands the arsenal of tools for rational design of DNA-based molecular devices with defined operational parameters. Although the presented system is an in vitro model, this approach (with appropriate adjustments for molecular crowding19) may eventually become extrapolated toward intracellular applications with the involvement of biochemically or clinically relevant enzymes (e.g. telomerase). The molecular simplicity and compact nature of the presented device should help circumvent concerns over its intracellular delivery. General compatibility of i-motif systems with nuclease resistant DNA backbone modification should in principle address concerns over the device stability in biological media.20 Programmability (a characteristic of DNA-based scaffolds) enables device adjustment for particular targeted conditions. Therefore, considering that i-motif based sensors have already been demonstrated to function inside cells2 and living systems,3 the present findings demonstrate potential of this approach to develop powerful tools for assessment of different conditions in biological environments.
Conclusions In conclusion, we have designed and evaluated a simple allosteric single-stranded DNA-based dual-input switch operating in physiologically relevant conditions. The switch utilizes intracellularlyrelevant triggers such as small change in pH and presence of polymerase activity as two inputs and synthesizes in situ a chemical output (oligonucleotide fragment) only when both inputs are present. It can be a viable alternative to molecular devices that rely on mRNA expressions or other indicators associated with redundant pathways, as well as devices which have an output moiety already incorporated as part of their molecular structure. We anticipate that the device architecture based on manipulating hairpin stability via inserted quadruplexed structures can be extended to confer other appropriate triggers.
Acknowledgements This work was supported by LSU College of Science. Purchase of CD spectrometer was funded by the National Science Foundation CRIF grant CHE-0840516.
Notes and references 1 C. Battle, X. Chu and J. Jayawickramarajah, Supramol. Chem., 2013, 25, 848; T. S. Bayer and C. D. Smolke, Nat. Biotechnol., 2005, 23, 337; A. R. Buskirk, A. Landrigan and D. R. Liu, Chem. Biol., 2004, 11, 1157.
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2 S. Modi, C. Nizak, S. Surana, S. Halder and Y. Krishnan, Nat. Nanotechnol., 2013, 8, 459; S. Modi, M. G. Swetha, D. Goswami, G. D. Gupta, S. Mayor and Y. Krishnan, Nat. Nanotechnol., 2009, 4, 325. 3 S. Surana, J. M. Bhat, S. P. Koushika and Y. Krishnan, Nat. Commun., 2011, 2, 7. 4 S. M. Douglas, I. Bachelet and G. M. Church, Science, 2012, 335, 831; Z. Z. Huang, J. S. Ren and X. G. Qu, Mol. BioSyst., 2012, 8, 921; J. Guo and R. Q. Yang, Mol. BioSyst., 2012, 8, 927. 5 F. Wang, C. H. Lu and I. Willner, Chem. Rev., 2014, 114, 2881; H. Pei, L. Liang, G. Yao, J. Li, Q. Huang and C. Fan, Angew. Chem., Int. Ed., 2012, 51, 9020; M. Leisner, L. Bleris, J. Lohmueller, X. Zhen and Y. Benenson, Nat. Nanotechnol., 2010, 5, 666; K. Rinaudo, L. Bleris, R. Maddamsetti, S. Subramanian, R. Weiss and Y. Benenson, Nat. Biotechnol., 2007, 25, 795; E. Shapiro and B. Gil, Science, 2008, 322, 387; Z. Xie, L. Wroblewska, L. Prochazka, R. Weiss and Y. Benenson, Science, 2011, 333, 1307; C. J. Delebecque, A. B. Lindner, P. A. Silver and F. A. Aldaye, Science, 2011, 333, 470; B. Gil, M. KahanHanum, N. Skirtenko, R. Adar and E. Shapiro, Nano Lett., 2011, 11, 2989; M. N. Win and C. D. Smolke, Science, 2008, 322, 456. 6 K. Watanabe, M. Harada-Shiba, A. Suzuki, R. Gokuden, R. Kurihara, Y. Sugao, T. Mori, Y. Katayama and T. Niidome, Mol. BioSyst., 2009, 5, 1306; M. Mahato, P. Kumar and A. K. Sharma, Mol. BioSyst., 2013, 9, 780; Acc. Chem. Res., Special Issue on Gene Silencing and Delivery, 2012, 45(7), 959–1172; L. Cui, J. L. Cohen, C. K. Chu, ´chet, J. Am. Chem. P. R. Wich, P. H. Kierstead and J. M. J. Fre Soc., 2012, 134, 15840; V. Sokolova and M. Epple, Angew. Chem., Int. Ed., 2008, 47, 1382; R. L. Juliano, X. Ming and O. Nakagawa, Bioconjugate Chem., 2012, 23, 147. 7 W. S. Dalton and S. H. Friend, Science, 2006, 312, 1165; D. Hanahan and R. A. Weinberg, Cell, 2011, 144, 646; E. E. Schadt, Nature, 2009, 461, 218. 8 A. V. Pinheiro, D. Han, W. M. Shih and H. Yan, Nat. Nanotechnol., 2011, 6, 763. 9 F. C. Simmel, Nanomedicine, 2007, 2, 817. 10 I. V. Nesterova, S. O. Elsiddieg and E. E. Nesterov, J. Phys. Chem. B, 2013, 117, 10115. 11 S. Ahmed, A. Kintanar and E. Henderson, Nat. Struct. Biol., 1994, 1, 83; K. Gehring, J. L. Leroy and M. Gueron, Nature, ´ron, J. L. Mergny and 1993, 363, 561; J. L. Leroy, M. Gue ´le `ne, Nucleic Acids Res., 1994, 22, 1600. C. He ´le `ne, 12 J. L. Mergny, L. Lacroix, X. G. Han, J. L. Leroy and C. He J. Am. Chem. Soc., 1995, 117, 8887. 13 Complete sequences of Ii, Ih, Is, p, i, and Target II are included in Table S1 in ESI†. 14 Details of experimental design and procedures are included in ESI†. 15 A. A. El-Harakany, F. M. Abdel Halim and A. O. Barakat, J. Electroanal. Chem. Interfacial Electrochem., 1984, 162, 285. 16 Available from: http://www.neb.com/nebecomm/products/ faqproductM0275.asp#180.
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17 I. V. Nesterova and E. E. Nesterov, J. Am. Chem. Soc., 2014, 136, 8843. 18 Y. Weizmann, M. K. Beissenhirtz, Z. Cheglakov, R. Nowarski, M. Kotler and I. Willner, Angew. Chem., Int. Ed., 2006, 45, 7384; A. R. Connolly and M. Trau, Angew. Chem., Int. Ed., 2010, 49, 2720. 19 A. Rajendran, S.-i. Nakano and N. Sugimoto, Chem. Commun., 2010, 46, 1299. 20 N. Kumar, M. Petersen and S. Maiti, Chem. Commun., 2009, 1532; C. C. Hardin, M. Corregan, B. A. Brown and L. N. Frederick, Biochemistry, 1993, 32, 5870; J. L. Mergny
2814 | Mol. BioSyst., 2014, 10, 2810--2814
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and L. Lacroix, Nucleic Acids Res., 1998, 26, 4797; H. Kanehara, M. Mizuguchi, K. Tajima, K. Kanaori and K. Makino, Biochemistry, 1997, 36, 1790; N. K. Sharma and K. N. Ganesh, Chem. Commun., 2005, 4330; Y. Krishnan-Ghosh, E. Stephens and S. Balasubramanian, Chem. Commun., 2005, 5278; J. A. Brazier, J. Fisher and R. Cosstick, Angew. Chem., Int. Ed., 2006, 45, 114; N. Kumar, J. T. Nielsen, S. Maiti and M. Petersen, Angew. Chem., Int. Ed., 2007, 46, 9220; C. P. Fenna, V. J. Wilkinson, J. R. P. Arnold, R. Cosstick and J. Fisher, Chem. Commun., 2008, 3567; Y. Zhao, L. Cao, J. Ouyang, M. Wang, K. Wang and X. H. Xia, Anal. Chem., 2013, 85, 1053.
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