ACS Synth Biol. 2017 May 19;6(5):758-765.

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

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

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

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

c Key Laboratory of Low-grade Energy Utilization Technologies & Systems of the Ministry of Education, Chongqing University, Chongqing, 40004, PR China

* Corresponding Authors: Lichun Dong and Ningzi Guan

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

Tel: +86-23-65106053

E-mail address: lcdong72@cqu.edu.cn (Lichun Dong); ningzi.guan@chbe.gatech.edu (Ningzi.Guan)

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

 

Abstract:

We describe here a novel gene regulation system to enhance the transcription of the target gene by introducing duplex aptamers upstream T7 promoter in the sense and antisense strands of DNA symmetrically. With the presence of thrombins, a DNA bubble would be formed around the aptamers in the local structure of DNA duplex due to the binding of aptamers with thrombins. Consequently, the recognition region of the promoter contained in the DNA bubble can be more easily recognized and bound by RNA polymerases, and the separation efficiency of the unwinding region of the promoter contained in the DNA bubble can also be significantly improved, leading to the enhanced expression of the target gene at the transcriptional level. 

DOI: 10.1021/acssynbio.6b00391 

 

Supplement

Artificial control of bio-functions through regulating gene expression is one of the most important and attractive technologies to build novel living systems useful for chemical synthesis, nanotechnology, pharmacology and cell biology.[1-4] The strategies to regulate gene expression through external stimulus, such as heat, electric field, pH and light, are restricted since a demanding growth environment is required for the growth of organisms.[5] It has been demonstrated that the regulators based on the synthetic biology technologies combined with function-encoding DNA molecules are effective to fundamentally control the expression of target genes.[6] For optimizing the gene expression by using these conventional methods, it is important to control the expression level of the individual gene for genetic circuits to operate at the desired level and achieve the ideal dynamic ranges, particularly regulating the toxic genes or those that may interfere with the host machinery.[7, 8] Accordingly, diverse molecular tools have been designed to regulate the gene expression at both the transcriptional level (e.g. constitutive/inducible promoters with high dynamic ranges)[9] and translational level (e.g. RNA-based regulators, libraries of ribosomal binding sites (RBSs)).[5] However, a large portion of gene regulation systems that were predicted theoretically to perform well execute poorly in practice, furthermore, it is also uncertain if the same gene regulation system could work well when being transferred to other species and environment since the current molecular tools are strictly specific in hosts and conditions.[5] Accordingly, a gene regulation system with the universal applicability is highly expected.

Here we describe a novel approach to enhance the expression of target gene at the transcriptional level by introducing duplex aptamers upstream T7 promoter in the sense and antisense strands of DNA symmetrically. With the presence of ligands, thrombins in this study, a DNA bubble would be formed around the duplex aptamers in the local structure of DNA duplex due to the binding of aptamers with thrombins (Figure 1a). Consequently, the recognition region of the promoter contained in the DNA bubble can be more easily recognized and bound by RNAPs, and the separation of the unwinding region of the promoter contained in the DNA bubble can also be accelerated, leading to the enhanced expression of the target gene at the transcriptional level. The DNA bubble in the approach is induced through a physical method based on the binding of DNA aptamers with the corresponding ligands, the mechanism is applicable for other microbes without considering the different specificities. However, the enhancing effect of the DNA bubble-mediated gene regulation is dependent on the distance between the duplex aptamers and the promoter (DBDAP),[6] which can be classified into the followed scenarios : i) when the distance is too long, both the sequences of the recognition region and the unwinding region cannot be contained in the induced DNA bubble, which would not play its function to enhance gene expression (Figure 1b); ii) when partial sequences of the recognition region are contained in the induced DNA bubble while no sequence of unwinding region is contained, the recognition capability of RNAP to the promoter can be strengthened while the separation efficiency of the unwinding region is not affected; consequently, the expression of target gene can be enhanced to some extent (Scheme 1b);[7] iii) when the sequences of the recognition region and partial sequences of the unwinding region are contained in the induced DNA bubble at a suitable range of DBDAP, both the recognition of RNAP to the promoter and the separation of the unwinding region can be improved, leading to the enhanced expression of the target genes to the maximum extent (Figure 1b); iv) when DBDAP is too short, although the sequences of both the recognition region and the unwinding region are contained in the DNA bubble, the recognition region of the promoter is presumably sequestered by the bound thrombins, resulting in the weakened  recognition of the recognition region by RNAP and a reduced expression of the target gene (Figure 1b).

The effect of the DNA bubble-mediated gene regulation system on the expression of gfp gene was investigated by introducing aptamers 33 bp upstream T7 promoter (DBDAP = 33 bp) (Figure 2a). At the situation without thrombins, the green fluorescent protein (GFP, encoded by gfp gene) concentration of templates with single or duplex aptamers exhibited no obvious difference compared with that without aptamer, demonstrating that the gene regulation system cannot play its role without the presence of thrombins. Moreover, at the situation with 1.2 μM thrombins, the GFP concentration of templates with single aptamer also exhibited insignificant change compared with that without aptamer, demonstrating that it has no effect on gene expression by introducing single aptamer upstream promoter in the sense or antisense strand of DNA even with the presence of thrombins. On the contrary, the GFP concentration of template with duplex aptamers exhibited a 50% increase, indicating that the introduction of duplex aptamers can significantly enhance the gene expression with the existence of thrombins. Subsequently, kinetic assays were performed on templates modified with duplex aptamers, single aptamer and none aptamer, respectively. According to the Michaelis-Menten plots of transcription rate versus the concentration T7 promoter (Figure 2b), [8, 9] Km and kcat for the three templates were calculated and showed in Figure 2b. Compared with that of template without aptamer, Km of the template with duplex aptamers significantly decreases while that of the template with single aptamer exhibited almost no difference, demonstrating that the introduction of duplex aptamers 33 bp upstream T7 promoter could strongly increase the recognition capability of RNAP to the promoter. On the other side, kcat of all the three templates is substantially same, indicating that the introduction of duplex aptamers 33bp upstream T7 promoter does not affect the separation efficiency of the unwinding region.

For evaluating the reliability of the constructed gene regulation system, the effect of the induced DNA bubble on the expression of ecaA gene was also studied by using enzyme assays. The results in Figure 3 showed that with the existence of 1.2 μM thrombins, the activity of carbonic anhydrase (CA) encoded by ecaA gene with duplex aptamers is almost 4-fold higher than that without the duplex aptamers, confirming that the developed DNA bubble-mediated gene regulation system is capable of enhancing gene expression. However, gene expression can be controlled at the transcriptional or the translational level;[10] moreover, the translational level may be improved by the induced DNA bubble and the amount of proteins would also be increased at this situation even if the conversion process from DNA to mRNA is not be regulated.[11-13] Therefore, the mechanism for the induced DNA bubble to affect gene expression should be further clarified, accordingly, the template containing the duplex aptamer-modified ecaA gene was investigated at the existence of 9 different concentrations of thrombins, in which, the expression of the ecaA gene was evaluated by using enzyme assays and the mRNA level was analysed by using the Real-time quantitative PCR. The results in Figure 3 showed that, at different concentrations of thrombins, the multiple of ecaA mRNA amplification is different as well as the activity of the ecaA gene, which is almost proportional to the multiple of ecaA mRNA amplification. These observations confirmed that the induced DNA bubble gene enhances the gene expression at the transcriptional level.

In conclusion, a novel regulation system for enhancing gene expression was constructed by introducing duplex aptamers upstream promoter in the two strands of DNA. The mechanism is applicable for other microbes without the necessary to consider the different specificities, therefore, the system can be extended to other bacterial or bacteriophagic promoter sequences, providing a general artificial method to control bio-functions through the powerful gene expression regulation tool.

 

 

Figure 1 A DNA bubble can be induced by introducing duplex aptamers upstream promoter with the existence of thrombins, which can enhance gene expression at the transcriptional level. (a) The DNA bubble can only be induced by introducing duplex aptamer upstream promoter and with the existence of specific ligand (thrombin 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.

 

 

 

Figure 2 Effect of the introduction of aptamer(s) upstream promoter on the expression of gfp gene. (a) GFP concentration of templates with no, single or duplex aptamers upstream promoter after 6 h reaction of cell-free protein synthesis experiments with/without the existence of 1.2 μM thrombins when DBDAP = 33 bp; (b) 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.

 

 

Figure 3 Effect of the aptamer-induced DNA bubble on the expression of ecaA gene and relationship between the multiple of mRNA amplification and enzymatic activity of the ecaA gene, a linear relationship can be built: the multiple of mRNA amplification n=0.82929 × CA activity/(U/mg)+0.05727, (R2=0.93816).

 

 

 

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