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Orientation controllable DNA biosensors hold great application potentials in recognizing small molecules and detecting DNA hybridization. Though electric field is usually used to control the orientation of DNA molecules, it is also of great importance and significance to seek for other triggered methods to control the DNA orientation. Here, we design a new strategy for controlling DNA orientation in biosensors. The main idea is to copolymerize DNA molecules with responsive polymers that can show swelling/deswelling transitions due to the change of external stimuli and then graft the copolymers onto an uncharged substrate. In order to highlight the responsive characteristic, we take thermo-responsive polymers as an example and reveal multi-responsive behavior and the underlying molecular mechanism of the DNA orientation by combining dissipative particle dynamics simulation and molecular theory. Since swelling/deswelling transitions can be also realized by using other stimuli-responsive (like pH and light) polymers, the present strategy is universal, which can enrich the methods of controlling DNA orientation and may assist with the design of the next generation of biosensors.
Dna Based Biosensors Pdf Download
Biosensors, which transduce a bio-recognition event into measurable electronic or opto-electronic signal, play a crucial role in a wide range of applications, including clinical diagnosis, environmental monitoring, forensic analysis and antiterrorism1,2,3,4,5. As one of the most important biosensors, double-stranded (ds) DNA sensors have been studied extensively by electrochemical methods, where controlling orientation is of great significance6,7. Previous results have shown that the orientation of end-tethered dsDNA on metal substrate can be controlled by alternating current (AC) electric field, while single-stranded DNA (ssDNA) cannot8,9. Based on the different behaviors of ssDNA and dsDNA, DNA hybridization can be identified10,11,12. Moreover, dsDNA orientation switching dynamics in electric field will become slow when bound with biomarkers, such as nucleic acids, small molecules, ions and proteins. The detection and size analysis of these biomarkers can thus be achieved on the basis of this property13,14,15,16.
In this work, we propose a new strategy to control the orientation of DNA molecules in biosensors, namely, by copolymerizing DNA molecules with responsive polymer and then grafting the copolymer onto an uncharged substrate. In particular, the polymer can undergo swelling/deswelling transitions via altering external stimulus strength. To illustrate the responsive property, here we use a general thermo-responsive polymer and PNIPAm in dissipative particle dynamics (DPD) simulation and molecular theory, respectively. As we will show below, the DNA orientation can show dual- and triple-thermo-responsive behaviors under different polymer lengths. Further, we will also reveal the underlying physical mechanism of the responsive behaviors in our new system.
In the present study, we propose a new strategy to control the DNA orientation in biosensors and combine DPD simulation and molecular theory to prove its feasibility. Especially, when the thermo-responsive polymer has proper length, the DNA orientation is triple-responsive as shown in Fig. 7. The order parameter is low at low temperature. As the temperature increases, the DNA molecules get much ordered. But further increase of temperature leads to the decrease of the DNA order parameter. Furthermore, we point out the feasibility and application of our reported strategy in real detections. Firstly, we should notice that the present strategy can also be used to detect the binding of small molecules. In order to demonstrate this, we also investigate the DNA orientation as a function of temperature in the presence of protein and find that the DNA order parameter becomes larger than that in the absence of protein (see Supplementary Figure 5). Due to the fact that the variation of DNA orientation can be detected by fluorescence energy transfer16, the detection of proteins may be achieved. Besides, when detecting charged molecules (e.g., proteins), the present strategy could be more suitable than electric-field method, since the electric field may confuse the binding of charged molecules. However, when compared with electric-field method, the variation degree of DNA orientation in our strategy is not so obvious, which may lead to a lower sensitivity of the detection. Moreover, small molecules may bind to thermo-responsive polymers32, which will also influence the sensitivity of the detection. These are beyond the scope of the present work and we will address the above issues in our future works.
Finally, we should notice that although thermo-responsive polymers are used in our study, the new strategy here is general because the responsive behavior of DNA orientation is just caused by swelling/deswelling transitions of polymers. Therefore, other types of stimuli-responsive polymers can also be used to take place of the thermo-responsive polymers in the system. For example, by using pH-sensitive polymers like PAH20,33, the pH-responsive detectiom can be fabricated, which may be well suited in specific organs (such as the gastrointestinal tract or the vagina) or intracellular compartments (such as endosomes or lysosomes); The light-responsive detection can be synthesized by using photo-sensitive polymer containing o-nitrobenzyl21,34, which may have the advantage of non-invasiveness and the possibility of remote spatiotemporal control. Therefore, our strategy here can enrich the methods of controlling DNA orientation. We expect that the new strategy for controlling DNA orientation could be engineered experimentally with the advance of present nanotechnology and believe that it can promote future development on novel design of biosensors.
Aptamers consist of short oligonucleotides that bind specific targets. They provide advantages over antibodies, including robustness, low cost, and reusability. Their chemical structure allows the insertion of reporter molecules and surface-binding agents in specific locations, which have been recently exploited for the development of aptamer-based biosensors and direct detection strategies. Mainstream use of these devices, however, still requires significant improvements in optimization for consistency and reproducibility. DNA aptamers are more stable than their RNA counterparts for biomedical applications but have the disadvantage of lacking the wide array of computational tools for RNA structural prediction. Here, we present the first approach to predict from sequence the three-dimensional structures of single stranded (ss) DNA required for aptamer applications, focusing explicitly on ssDNA hairpins. The approach consists of a pipeline that integrates sequentially building ssDNA secondary structure from sequence, constructing equivalent 3D ssRNA models, transforming the 3D ssRNA models into ssDNA 3D structures, and refining the resulting ssDNA 3D structures. Through this pipeline, our approach faithfully predicts the representative structures available in the Nucleic Acid Database and Protein Data Bank databases. Our results, thus, open up a much-needed avenue for integrating DNA in the computational analysis and design of aptamer-based biosensors.
The relatively simple chemical structure of aptamers allows the insertion of electrochemical or fluorescent reporter molecules7 as well as surface-binding agents8 in specific locations on the oligonucleotide9. During probe-target binding, the conformation change of the aptamer may be exploited to generate an analytical signal10. A number of aptamer-based biosensors have been successfully used to measure cell secretion of proteins11, 12; however, several opportunities for improvement remain before commercialization is feasible13. These include enhancing the sensitivity and improving the manufacturability and repeatability of aptamer-based sensors. Both of these challenges are not easily overcome without a better understanding of the molecular level interactions of the aptamer-biosensor surface (for improving manufacturability, reproducibility, and sensitivity) as well as of the aptamer-protein complex (for improving specificity and sensitivity).
Here, we present the first approach to predict the three-dimensional structures of single stranded DNA required for aptamer applications that extends current sequence-based computational efforts for RNA18,19,20,21. Our approach faithfully predicts the representative resolved structures available in the Nucleic Acid Database (NDB) and Protein Data Bank (PDB) databases from a pipeline that integrates 2D and 3D structural tools, including Mfold, Assemble 2, Chimera, VMD, and Molecular Dynamics (MD) simulations. Explicitly, we build ssDNA secondary structure from sequence, construct equivalent 3D ssRNA models, transform the 3D ssRNA models into ssDNA 3D structures, and finally refine the resulting ssDNA 3D structures through energy minimization. To thoroughly evaluate our approach, we considered all hairpin-like ssDNA molecules with experimentally solved 3D structures selected through an exhaustive search for ssDNA molecules and aptamers in the PDB database. Our results indicate that this approach works exceptionally well for the hairpin-like structural motif of ssDNA, the focus of the current work. To test the robustness of the results, we performed additional atomistic MD simulations for a sub-set of representative ssDNA molecules and aptamers. The atomistic details available in MD simulations with explicit solvent have been fundamental at uncovering the molecular level mechanisms of key experimental observations and deepen our understanding of the interactions and properties of biological complexes in their natural environment24,25,26,27,28. Partly because of the lack of solved 3D structures, very few MD simulation studies have focused on aptamers29, 30. Our results show that MD simulations can, indeed, be used to further improve the structural predictions and that the predictions are representative of those obtained from the dynamics of the systems under conditions that mimic their targeted environment. 350c69d7ab