A DNA Bubble-Mediated Gene Regulation System Based on

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Letter

A DNA bubble-mediated gene regulation system based on thrombin-bound DNA aptamers Jing Wang, Le Yang, Xun Cui, Zhe Zhang, Lichun Dong, and Ningzi Guan ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00391 • Publication Date (Web): 01 Feb 2017 Downloaded from http://pubs.acs.org on February 3, 2017

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ACS Synthetic Biology

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A DNA bubble-mediated gene regulation system based on thrombin-bound DNA aptamers

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Jing Wang a, b,c †, Le Yang a, b,c †, Xun Cui a, Zhe Zhang b, Lichun Dong *a,c, Ningzi Guan *b

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a

School of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044, China

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b

School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, 30332, USA

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c

Key Laboratory of Low-grade Energy Utilization Technologies & Systems of the Ministry of Education, Chongqing

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University, Chongqing, 40004, PR China

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* Corresponding Authors: Lichun Dong and Ningzi Guan

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† Co-first Authors: Jing Wang and Le Yang contributed equally to this work

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Tel: +86-23-65106053

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E-mail address: [email protected] (Lichun Dong); [email protected] (Ningzi.Guan)

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Mailing address: 3137 MoSE, Georgia Institute of Technology, North Avenue, Atlanta, GA, 30332.

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ACS Paragon Plus Environment


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Abstract: We describe here a novel approach to enhance the transcription of target gene in cell-free systems by

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symmetrically introducing duplex aptamers upstream to T7 promoter in both the sense and antisense strands of

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double-stranded plasmids, which leads to the formation of a DNA bubble due to the none-complementary state of

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the ssDNA region harboring the aptamer sequences. With the presence of thrombins, the DNA bubble would be

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enlarged due to the binding of aptamers with thrombins. Consequently, the recognition region of the promoter

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contained in the DNA bubble can be more easily recognized and bound by RNA polymerases, and the separation

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efficiency of the unwinding region can also be significantly improved, leading to the enhanced expression of the

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target gene at the transcriptional level. The effectiveness of the proposed gene regulation system was demonstrated

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by enhancing the expression of gfp and ecaA genes in cell-free systems.

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For Table of Contents use only

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A DNA bubble-mediated gene regulation system based on thrombin-bound DNA aptamers

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Jing Wang a, b, c, †, Le Yang a, b, †, Xun Cui a, Zhe Zhang b, Lichun Dong *a,c, Ningzi Guan *b

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Keywords: gene expression regulation• DNA bubble• aptamer• promoter

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Artificial control of bio-functions through regulating gene expression is one of the most important and attractive

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technologies to build novel living systems useful in the areas of chemical synthesis, nanotechnology, pharmacology,

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cell biology, etc.1-4 The strategies to regulate gene expression through external stimulus, such as heat, electric field,

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pH and light, are restricted since a demanding growth environment is required for the growth of organisms.5 It has

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also been well demonstrated that the regulators by deploying synthetic biology technologies with function-encoding

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DNA molecules are effective to fundamentally control the expression of target genes.6 For optimizing gene

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expression by using these conventional methods, it is important to control the expression of the individual genes for

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genetic circuits to operate at the desired level and achieve the ideal dynamic ranges, particularly regulating the toxic

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genes or those that may interfere with the host machinery.7, 8 Accordingly, various molecular tools have been

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designed to regulate gene expression at both the transcriptional level (e.g. constitutive/inducible promoters with high

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dynamic ranges)9 and translational level (e.g. RNA-based regulators, libraries of ribosomal binding sites (RBSs)).5

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However, a large portion of gene regulation systems, which were theoretically predicted to perform well, executed

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poorly in practice; furthermore, it is also uncertain whether the same gene regulation system could work well when

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being transferred to other species or environments since the current molecular tools are strictly specific in hosts and

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conditions.5 Accordingly, a gene regulation system with the universal applicability is highly expected.

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It is well recognized that the transcription is initiated with the formation of RNA polymerase (RNAP)-promoter

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complex via the binding of RNAP to the recognition region of the promoter, which is followed by the DNA strand

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separation through melting of the unwinding region of the promoter. Then, the promoter-bound RNAP polymerizes

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the first few nucleotides (up to 10). After the transcription is initiated, the transcript becomes long enough to form a

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stable hybrid with the template strand, helping to stabilize the transcription complex. Subsequently, RNAP changes

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to its elongation conformation, loses its σ-factor and moves away from the promoter. Among the transcription

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process, the formation of RNAP-promoter complex and separation of the unwinding region are the two rate-limiting

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steps.10 Therefore, the expression of a target gene can be potentially enhanced by strengthening the recognition

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capability of RNAP to the recognition region and speeding up the separation of the unwinding region. In this sense,

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nucleic acid aptamers, which include DNA aptamer (single-stranded DNA molecule) and RNA aptamer (single-

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stranded DNA molecule) that can be engineered to bind to specific molecules tightly,11 could be utilized to realize

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the potential strategies. Accordingly, RNA aptamers have been extensively used to regulate the transcription or

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translation of target genes, often by coupling with the binding event and ensuing the conformational change.12,13

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Nucleic aptamers can be chosen against any ligands of interest from a combinatorial library by using an iterative

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affinity selection procedure, in addition, the current hybridization rules can facilitate the predictive and rational

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design of nucleic acid domains,11,14 which endows the aptamer-based gene regulation systems the ability to regulate

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gene expression for a wide variety of species through a simple method.15

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In a previous study,15 an approach by using aptamers to repress gene expression at the transcriptional level has

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been described by placing a thrombin-bound ssDNA aptamer downstream to the T7 promoter. In the absence of 4


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thrombin, T7 RNA polymerases are able to transcribe the templates. Once the DNA aptamer was bound by thrombins,

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the transcription from the T7-aptamer promoter can be repressed. On the contrary, the transcriptional level of the

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target gene is enhanced in this study by symmetrically introducing duplex aptamers (two sequences of same ssDNA

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aptamers) upstream to the T7 promoter in the sense and antisense strands of a double-stranded plasmid, respectively,

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which leads to the formation of a DNA bubble due to the none-complementary state of the DNA aptamer.15 With the

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presence of ligands (thrombins in this study), the DNA bubble would be enlarged around the duplex aptamers in the

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local structure of the double-stranded plasmid due to the binding of aptamers with thrombins. Consequently, the

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recognition region of promoter contained in the enlarged DNA bubble can be more easily recognized and bound by

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RNAPs, and the separation of the unwinding region of promoter contained in the DNA bubble can also be accelerated,

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leading to the enhanced expression of the target gene at the transcriptional level. The DNA bubble in this approach

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is manipulated through a physical method based on the binding of DNA aptamers with its corresponding ligands, the

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mechanism is generally applicable for other microbes without considering the different specificities. However, the

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enhancing effect of the DNA bubble-mediated gene regulation is dependent on the distance between the duplex

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aptamers with the promoter (DBDAP),16 which can be classified into the followed scenarios: i) when the distance is

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too long, both the sequences of the recognition region and the unwinding region cannot be contained in the induced

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DNA bubble, whose function to enhance gene expression cannot be fulfilled (Figure 1); ii) when partial sequences

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of the recognition region are contained in the induced DNA bubble while no sequence of unwinding region is

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contained, the recognition capability of RNAP to the promoter can be strengthened while the separation efficiency

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of the unwinding region is not affected; the expression of the target gene can be enhanced to some extent (Figure

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1);17 iii) when the sequences of the recognition region and partial sequences of the unwinding region are contained

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in the induced DNA bubble at a suitable range of DBDAP, both the recognition of RNAP to the promoter and the

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separation efficiency of the unwinding region can be improved, leading to the enhanced expression of the target gene

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to the maximum extent (Figure 1); iv) when DBDAP is too short, although the sequences of both the recognition

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region and unwinding region are contained in the DNA bubble, the recognition region of the promoter is presumably

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sequestered by the bound thrombins, resulting in the weakened recognition of the recognition region by RNAP and

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a reduced expression of the target gene (Figure 1).

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For demonstrating the effectiveness of the DNA bubble-mediated gene regulation, the expression of gfp gene was

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investigated by introducing a single or duplex aptamers 12bp upstream to T7 promoter (DBDAP = 12 bp) (Figure

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2a-2b) in a double-stranded plasmid containing gfp gene in a cell-free system. At the situation without thrombins,

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the green fluorescent protein (GFP, encoded by gfp gene) concentration of the template introduced with a single

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aptamer (ssDNA) in one strand (sense or antisense) or duplex aptamers (two ssDNA aptamers) in both the strands

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of the double-stranded plasmid (Figure S1, Figure S2) exhibited no obvious difference compared with the template

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with no aptamer being introduced in the plasmid (Figure 2a-2b), demonstrating that the proposed gene regulation

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system cannot affect the expression of gfp gene without the presence of thrombins. Moreover, in the presence of 1.2

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µM of thrombins, GFP concentration of the template introduced with a single aptamer in one strand also exhibited

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no significant change compared with that of the template with no aptamer being introduced (Figure 2a-2b), indicating 5



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that it has no effect on gene expression by introducing a single aptamer upstream to promoter in one strand of the

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double-stranded plasmid even with the presence of thrombins. On the contrary, GFP concentration of template with

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duplex aptamers being introduced in the double-stranded plasmid exhibited a 50% increase compared with that of

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the template with no aptamer being introduced (Figure 2a-2b), clarifying that the introduction of duplex aptamers in

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both the sense and antisense strands of the double-stranded plasmid can significantly enhance the gene expression

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with the presence of thrombins. Subsequently, kinetic assays were performed on templates introduced with duplex

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aptamers, a single aptamer and no aptamer, respectively. According to the Michaelis-Menten plots of transcription

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rate versus concentration of T7 promoter (Figure 2c),18,19 Km and kcat for the three kinds of templates were calculated

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and showed in Figure 2c. Compared with that of template without aptamer being introduced, Km of the template

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introduced with duplex aptamers 12bp exhibited a significantly decrease while that of the template introduced with

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a single aptamer 12bp showed no obvious difference, demonstrating that the introduction of duplex aptamers 12bp

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upstream to T7 promoter can significantly enhance the recognition capability of RNAP to the promoter. On the other

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side, kcat of the template introduced with duplex aptamers significantly increased while that of the template

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introduced with a single aptamer exhibited undistinguished difference compared with that of the template without

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aptamer being introduced. Liu et al.16 has proposed a strategy for photo-regulation of gene expression with the

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azobenzene-tethered DNA, azobenzene is introduced into the promoter region, and the transcription reaction by

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RNAP is photo-regulated by trans-cis isomerization of the incorporated azobenzene. Their study found that tethering

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an azobenzene at the specific recognition region of a promoter can strongly enhance the binding of RNAP without

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changing the transcription rate constant (NTP), but tethering an azobenzene at the specific unwinding region

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enhanced the NTP without changing the affinity of RNAP to promoter. In this study, the recognition region of the

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promoter contained in the DNA bubble can be more easily recognized and bound by RNAPs and the separation of

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the unwinding region of the promoter contained in the DNA bubble can also be accelerated (corresponding to a larger

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NTP), leading to the enhanced expression of the target gene at the transcriptional level. The advantage of our strategy

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is that the recognition of RNAP to promoter and separation of the unwinding region can be enhanced at the same

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time, demonstrated by the decreased Km value and increased kcat value. The effect of the gene regulation system on

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the expression of gfp gene was also investigated for the cases of no aptamer, a single aptamer and duplex aptamers

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33bp (DBDAP = 33 bp) being introduced upstream to T7 promoter, the results showed that the Km value for the

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template introduced with duplex aptamers 33bp was decreased, while the kcat values of all the three kinds templates

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were substantially same (Figure S13), indicating that the introduction of duplex aptamers 33bp upstream to T7

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promoter can enhance the recognition of RNAP to the promoter, but does not affect the separation efficiency of the

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unwinding region.

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For clarifying the effect of DBDAP on gene expression, kinetic assays were performed on templates introduced

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with duplex aptamers of different DBDAPs. Figure 3a demonstrated that when DBDAP is less than 9 bp, a relatively

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larger Km would be obtained since the recognition region of the promoter is sequestered by the bound thrombins.

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When DBDAP is larger than 9 bp, the recognition region starts to be exposed from the bound thrombins, and can be

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completely exposed when DBDAP = 12 bp, at which, the Km value reaches the minimum. Afterwards, with a further 6


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increase in DBDAP, Km slightly increases with the recognition region moving out from the induced DNA bubble

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gradually until DBDAP = 39 bp, at which, no sequence of the recognition region is still contained in the induced

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DNA bubble. Figure 3b showed that when DBDAP < 33 bp, the kcat value increases with a decrease in DBDAP,

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meaning that the separation efficiency of the unwinding region can be improved by decreasing DBDAP. At these

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cases, partial sequences of the unwinding region can be contained in the induced DNA bubble while a smaller

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DBDAP indicates that more sequences would be contained. Figure 3c demonstrated the variation of GFP

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concentration for templates introduced with duplex aptamers at different DBDAPs in the presence of 1.2 µM

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thrombins. When DBDAP > 39 bp, no sequence of the promoter is contained in the induced DNA bubble, the

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expression of gfp gene would not be affected by the induced DNA bubble, resulting in a very low GFP concentration.

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When 12 bp < DBDAP < 39 bp, GFD concentration increases with a decrease in DBDAP, indicating that the

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enhancing effect of the induced DNA bubble on the expression of gfp gene becomes stronger at a smaller DBDAP.

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At this case, the sequences of the recognition region and the unwinding region start to be contained in the induced

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DNA bubble when DBDAP = 39 and 33 bp, respectively. The improvement of both the recognition of RNAP to the

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promoter and the separation efficiency of DNA strands with the unwinding region bring about the pronounced

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enhancement of gfp gene expression. While when DBDAP < 12 bp, the recognition region starts to be sequestered

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by the bound thrombins; the expression of gfp gene decreases sharply with a further decrease in DBDAP. When

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DBDAP < 9 bp, the recognition region of the promoter is completely sequestered by the bound thrombins, the

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expression of gfp gene reaches the lowest level, indicated by the minimum GFP concentration. In general, the

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optimum DBDAP for the DNA bubble-mediated gene regulation system to enhance the expression of gfp gene under

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T7 promoter is 12 bp. Under this condition, Km of the corresponding template introduced with the duplex aptamers

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in the presence of 1.2 µM of thrombin is 0.21, which is much lower than that without introducing the aptamer (0.74).

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On the other hand, kcat of the corresponding template introduced with the duplex aptamers in the presence of 1.2 µM

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of thrombin is 0.38, much higher than that without introducing the aptamer (0.26).

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For evaluating the reliability of the constructed gene regulation system, the effect of the induced DNA bubble on

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the expression of ecaA gene was also studied by using enzyme assays. The results in Figure 4a showed that with the

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existence of 1.2 µM thrombins, the activity of carbonic anhydrase (CA) encoded by ecaA gene introduced with

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duplex aptamers is almost 4-fold higher than that without introducing the aptamer, confirming that the proposed

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DNA bubble-mediated gene regulation system is capable of enhancing gene expression. However, gene expression

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can be controlled at both the transcriptional and the translational level;20 moreover, when the translational level of

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gene expression is improved by the induced DNA bubble, the amount of proteins would also increase even if the

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conversion process from DNA to mRNA is not be regulated.21-23 Therefore, the mechanism for the induced DNA

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bubble to affect gene expression should be further clarified, accordingly, the template containing the duplex aptamer-

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introduced ecaA gene was investigated at the existence of 9 different concentrations of thrombins, in which, the

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expression of ecaA gene was evaluated by using enzyme assays and the mRNA level was analyzed by using the

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Real-time quantitative PCR. The results in Figure 4b showed that, at different concentrations of thrombins, the

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multiple of ecaA mRNA amplification is different as well as the activity of ecaA gene, which is almost proportional 7



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to the multiple of ecaA mRNA amplification. These observations confirmed that the induced DNA bubble gene

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enhances the gene expression at the transcriptional level.

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In conclusion, a novel regulation system to enhance gene expression was constructed by symmetrically

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introducing duplex aptamers upstream to T7 promoter in the sense and antisense strands of a double-stranded plasmid,

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which leads to the formation of a DNA bubble. With the presence of thrombins, the DNA bubble would be enlarged

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around the duplex aptamers due to the binding of aptamers with thrombins. Consequently, once the recognition

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region and the unwinding region of promoter are contained in the DNA bubble, the recognition region can be more

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easily recognized and bound by RNAP, and the separation efficiency of the unwinding region can also be

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significantly improved, leading to the enhanced expression of the target gene at the transcriptional level.

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The DNA bubble in the proposed gene regulation system is induced through the none-complementary sequences

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of DNA aptamer and enlarged due to the binding of aptamers with thrombins.15 The method is generic and can be

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extended to other DNA aptamers and corresponding ligands. For example, the unwinding of the two functional

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regions of promoter (recognition region and unwinding region) could be potentially achieved by using helicases and

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dCas9, especially dCas9 to an activator domain, because the RNA-guided DNA-binding protein dCas9 is an

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excellent alternative candidate for unwinding specific region of DNA.28 In this case, the none-complementary

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sequences of ligand-bound aptamers do not need to be constructed as dCas9-based activator domain would induce

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the separation of complementary dsDNA without the need of using any fusion.

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Javaherian ea al.27 has introduced a method for developing specific aptamers for target proteins in crude cell lysate

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and purification of the target proteins from the cell lysate using the obtained aptamers. Through their approach, we

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can develop aptamers corresponding to specific proteins in future works, and construct duplex aptamer-introduced

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plasmids containing specific genes in living cells. Consequently, the metabolic flux of target products can be

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improved by overexpressing the key genes and binding the side-effect proteins using the gene regulation system

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developed in this study.

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Methods

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Construction of plasmids.

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To establish the aptamer-based gene regulation system, three kinds of double-stranded plasmids were constructed

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according to the standard techniques by cloning gfp gene with duplex DNA aptamers upstream to T7 promoter, a

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single DNA aptamer upstream to T7 promoter and a fully double-stranded T7 promoter without introducing aptamer

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into pET28a(+) vector backbones, respectively (Figure S1). Two double-stranded plasmids were constructed

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according to the standard techniques by cloning ecaA gene with duplex DNA aptamers upstream to T7 promoter and

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a fully double-stranded T7 promoter without introducing aptamer into pET28a(+) vector backbones, respectively.

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(Figure S1).

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All the plasmids, primers and strains used in this study are list in Table S1. The 5 fragments of the modified gfp

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or ecaA genes were obtained through the method of overlap PCR (Figure S3-Figure S12). Their backbones were

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generated by digesting pET28a(+) plasmid with XbaI and XhoI endonucleases, then the 5 products were isolated and 8


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purified. The backbone and 5 products were joined respectively using T4 ligase to give 5 parent plasmids:

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pET28aNgfp (no aptamer modified gpf gene as a control), pET28aSgfp, pET28aDgfp, pET28aNecaA (no aptamer

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modified gpf gene as a control) and pET28aDecaA.

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The aptamer-introduced gene regulation system was tested by incubating the DNA templates with thrombins along

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with 0.01% Tween-20 for 1 h at room temperature, followed by the addition of the cell extract. The used thrombins

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(purchased from MP Biomedicals, USA) were prepared by diluting the stock solution (in 50% glycerol) into 10 mM

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of Tris-HCl pH 7.5 and 50 mM of KCl to ensure that the final glycerol concentration in the cell-free protein synthesis

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reactions is less than 0.5%. The thrombin DNA aptamer sequences in the study used to enhance gene expression is

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5’-GGTTGGTGTGGTTGG-3’ by referring to the study of Iyer and Doktycz.15

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Cell-Free Protein Synthesis Experiments and GFP assays.

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The Promega S30 T7 High-Yield Expression System kit (Promega TM306) was used for the cell-free protein

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synthesis experiments. The S30 premix and the cell extract were mixed in the proportion recommended by the

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manufacturer, and templates were used at 25 ng/µL concentration per reaction. DNA concentrations used were based

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on Karig’s method 29. Reactions were set up in Corning CLS3820 plates following manufacturer’s instructions. For

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the GFP assays, the synthesis reactions of cell-free proteins were prepared firstly, incubated at 30 °C with shaking

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for 6 h, and fluorescence measurements (485/20 nm excitation, 528/20 nm emission) were made every 7 min in a

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Biotek Synergy 2 plate reader. Values indicated in the graphs representing GFP concentrations were obtained after 6

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h of synthesis reactions. Thrombin-dependent gene enhancement system was tested by incubating the DNA templates

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with thrombins along with 0.01% Tween-20 for 1 h at room temperature, followed by the addition of the cell extract.

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The used thrombins (purchased from MP Biomedicals, USA) were prepared by diluting the stock solution (in 50%

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glycerol) into 10 mM Tris-HCl pH 7.5 and 50 mM KCl to ensure that the final glycerol concentration in the cell-free

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protein synthesis reactions is less than 0.5%.

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Michaelis-Menten plots

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The operating conditions for the T7 RNAP reaction are as follows: [T7 RNAP (from TaKaRa)] = 50 unit in 20 µL

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(corresponding to 0.15 µM), [[α-32P]ATP] = 2 µCi in 20 µL, [each NTP] = 0.5 mM, [each strand of the promoter] =

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2.0 µM, and [spermidine] = 2 mM. Tris-HCl buffer (40 mM, pH 8.0) containing dithiothreitol (5 mM), MgCl2 (24

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mM), and NaCl (2 mM). First, a mixture of template and non-template strands were annealed in 10 mM of Tris-HCl

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buffer (pH 8.0) with 10 mM of NaCl by heating at 95 °C for 3 min and cooling to 37 °C for 30 min. Subsequently,

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the mixture was cooled in ice, and the stock solution involving NTPs and [α-32P] ATP was added. After T7 RNAP

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was added, the reaction mixture was incubated at 37 °C for 6 h to achieve the transcription. During the reaction, the

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mixture was sampled at a desired interval, and the transcription was stopped by adding dye solution containing 80%

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formamide, 50 mM EDTA, and 0.025% bromophenol blue (with the same volume as the sampled reaction mixture).

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For the Michaelis-Menten analysis, the transcription was carried out by changing promoter concentration. The ATP

9



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concentration was measured every 10 minutes by Microbial ATP ELISA Kit. In this experiment, the concentration

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of T7 RNA polymerase was kept constant at 0.20 µM.

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mRNA analysis and Enzyme assays.

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Messenger RNA analysis was performed by RT-PCR and real-time PCR. Total RNA was isolated using an E.Z.N.A

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total RNA kit I (Omega), 10 µg of total RNA was reversely transcribed with reverse transcriptase (Vazyme) to obtain

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the cDNA. Real-time PCR was performed in an ICycler (BioRad) monitoring double-stranded DNA assays

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continuously with SYBR-Green (Invitrogen). The fragments of the target gene in the diluted cDNA were amplified

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in a PCR with AmpliTaq Gold by the primer sets: CA-RT-up, CA-RT-Down; The Ct values of the target gene were

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compared to those of ecaA gene contained in the pET28a(+) plasmid to test the expression of the target gene on the

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transcriptional level.

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To analyze enzyme activities, the reaction liquid was centrifuged at 10000 g at 4 °C for 20 min and the resulted

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supernatant was used for enzyme activity assays. CA activity was expressed in Wilbur- Anderson units per mg of

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protein and was calculated using the formula [(t0/t-1) ×10]/mg protein, where t0 and t represent the time required for

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the pH to change from 8.0 to 7.0 in a buffer control and cell extracts, respectively.30

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Supporting Information

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All the plasmids, primers and strains used in this study are list in Table S1, plasmids construction are showed in

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supporting information (Figure S1). The 5 fragments of the modified gfp or ecaA genes were obtained through the

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method of overlap PCR (Figure S2-S12). Effect of the introduction of aptamer(s) 33 bp upstream to promoter on the

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expression of gfp gene (Figure S13). This information is available via the Internet at http://pubs.acs.org.

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Funding

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This work is financially supported by the National Science Foundation of China (51272296) and the graduate

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scientific research and innovation foundation of Chongqing, China (CYS16014).

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Authors’ contributions

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JW and LY are contributed equally to this work (performed the experiments), XC and ZZ revised the manuscript,

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LCD and NZG designed the work, analyzed the data, and drafted the manuscript. All authors read and approved the

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final manuscript.

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Figure 1 A DNA bubble can be induced by introducing duplex aptamers upstream to promoter, with the presence of thrombins, the gene expression at the transcriptional level can be enhanced. (a) The DNA bubble can only be induced by introducing duplex aptamer upstream to promoter and with the existence of specific ligands (thrombins in this study). (b) The effect of the DNA bubble-mediated gene regulation is dependent on the distance between the duplex aptamers and the promoter (DBDAP) i) when the distance is too long, the regulation system would not play its function to enhance gene expression; ii) the expression of target gene can be enhanced to some extent at a little long DBDAP; iii) the expression of the target gene can be enhanced to the maximum extent at a suitable DBDAP; iv) when DBDAP is too short, the recognition region of the promoter is sequestered by the bound thrombins, resulting in a weakened expression of the target gene. 13



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Figure 2 Effect of the introduction of aptamer(s) 12 bp upstream to promoter on the expression of gfp gene. (a) GFP concentration of templates introduced with no, single or duplex aptamers 12 bp upstream to promoter after 6 h reaction of cell-free protein synthesis experiments with/without the existence of 1.2 µM thrombins (DBDAP = 12 bp); (b) Kinetics of GFP concentration of templates introduced with no, single or duplex aptamers 12 bp upstream to promoter during 6 h reaction of cell-free protein synthesis experiments with/without the existence of 1.2 µM thrombins (DBDAP = 12 bp); (c) Michaelis-Menten plots of the transcription rate versus T7 promoter concentration under 0.20 µM T7 RNA polymerase. The ATP incorporation was used to describe the transcription rate.

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Figure 3 Effect of DBDAP (indicated by the base numbers between the promoter and aptamer) on gene expression. (a) Km of Michaelis-Menten plots (transcription rate versus T7

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promoter concentration under 0.20 µM T7 RNA polymerase. The ATP incorporation was used to describe the transcription rate under T7 RNA polymerase (0.20 µM) for the modified

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T7 promoters with different DBDAPs; (b) Kcal of Michaelis-Menten plots (transcription rate versus T7 promoter concentration under 0.20 µM T7 RNA polymerase. The ATP incorporation

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was used to describe the transcription rate) under T7 RNA polymerase (0.20 µM) for the modified T7 promoters with different DBDAPs; (c) GFP concentration as a function of DBDAP.

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Figure 4 Effect of the aptamer-induced DNA bubble on the expression of ecaA gene. (a) Effect of thrombin concentration on carbonic anhydrase (CA) activity of the duplex aptamers-

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modified ecaA gene (DBDAP = 12 bp). (b) Relationship between the multiple of mRNA amplification and the enzymatic activity, a linear relationship can be built: the multiple of mRNA

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amplification n=0.82929 × CA activity/(U/mg)+0.05727, (R2=0.93816).

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A DNA Bubble-Mediated Gene Regulation System Based on

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