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1.
J Am Chem Soc ; 2020 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-32453556

RESUMO

Genomic DNA is compacted via chromatin condensation in mammalian cells, and transcription of such topologically constrained DNA to message RNA is under strict spatiotemporal regulation. Nevertheless, control of DNA topology has been poorly explored in in-vitro transcription and gene transfection. Here we report the construction of topologically ordered (TO-) prokaryotic genes comprised of linear DNA templates appended with a T7 promoter sequence with the use of DNA self-assembly. We find that TO-DNA maintains the transcription activity whereas the activity is critically dependent on the configuration of the T7 promoter in a folded DNA nanostructure. By prescribing the position and the intactness of the T7 promoter, we can dynamically activate or repress transcription in response to specific DNA key strands in a Boolean logic manner. Bioorthogonal switchable transcription is realized with the insertion of multiple genes in a TO-DNA. Further, implementing TO-DNA in living bacterias leads to switchable transcription of fluorescent RNA aptamers for light-up cell imaging. Hence, the design of TO-DNAs provides a means for shape-dependent gene delivery, enriching the toolbox of genetic engineering and synthetic biology.

2.
Artigo em Inglês | MEDLINE | ID: mdl-32367682

RESUMO

Tumor progressions such as metastasis are complicated events that involve abnormal expression of different miRNAs and enzymes. Monitoring these biomolecules in live cells with computational DNA nanotechnology may enable discrimination of tumor progression via digital outputs. Herein, we report intracellular entropy-driven multivalent DNA circuits to implement multi-bit computing for simultaneous analysis of intracellular telomerase and microRNAs including miR-21 and miR-31. These three biomolecules can trigger respective DNA strand displacement recycling reactions for signal amplification. They are visualized by fluorescence imaging, and their signal outputs are encoded as multi-bit binary codes for different cell types. The results can discriminate non-tumorigenic, malignant and metastatic breast cells as well as respective tumors. This DNA computing circuit is further performed in a microfluidic chip to differentiate rare co-cultured cells, which holds a potential for the analysis of clinical samples.

3.
Artigo em Inglês | MEDLINE | ID: mdl-32395895

RESUMO

Artificial antigen presenting cells (APCs) with surface-anchored T cell activating ligands hold great potential in adoptive immunotherapy. However, it remains challenging to precisely control the ligand positioning on those platforms via conventional bioconjugation chemistry. Here, utilizing DNA-assisted bottom-up self-assembly, we are able to precisely control both lateral and vertical distribution of T cell activation ligands on red blood cells (RBCs). It is found that the clustered lateral positioning of peptide-major histocompatibility complex (pMHC) on RBCs with a short vertical distance to the cell membrane would be favorable for more effective T cell activation, likely owing to their better mimic of natural APCs. Such optimized RBC-based artificial APCs can stimulate T cell proliferation in vivo and effectively inhibit tumor growth via adoptive immunotherapy. DNA technology is thus a unique tool to precisely engineer the cell membrane interface and tune cell-cell interactions, promising for a wide range of applications including immunotherapy.

4.
ACS Nano ; 2020 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-32407064

RESUMO

DNA methylation is one of the principal epigenetic mechanisms that control gene expression in humans, and its profiling provides critical information about health and disease. Current profiling methods require chemical modification of bases followed by sequencing, which is expensive and time-consuming. Here, we report a direct and rapid determination of DNA methylation using an electric biosensor. The device consists of a DNA-tweezer probe integrated on a graphene field-effect transistor for label-free, highly sensitive, and specific methylation profiling. The device performance was evaluated with a target DNA that harbors a sequence of the methylguanine-DNA methyltransferase (MGMT), promoter of glioblastoma multiforme, a lethal brain tumor. The results show that we successfully profiled the methylated and non-methylated forms at picomolar (pM) concentrations. Further, fluorescence kinetics and molecular dynamics simulations revealed that the position of the methylation site(s), their proximity, and accessibility to the toe-hold region of the tweezer probe, are the primary determinants of the device performance.

5.
J Am Chem Soc ; 2020 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-32369359

RESUMO

Intracellular DNA-based hybridization reactions generally occur under tension rather than in free states, which are spatiotemporally controlled in physiological conditions. However, how nanomechanical forces affect DNA hybridization efficiencies in in-vitro DNA assays, for example, biosensors or biochips, remains largely elusive. Here, we design DNA framework-based nanomechanical handles that can control the stretching states of DNA molecules. Using a pair of tetrahedral DNA framework (TDF) nanostructured handles, we develop bridge DNA sensors that can capture target DNA with ultrafast speed and high efficiency. We find that the rigid TDF handles bind two ends of a single-stranded DNA (ssDNA) and hold it in a stretched state, with an apparent stretching length comparable to its counterpart of double-stranded DNA (dsDNA) via atomic force microscopy measurement. The DNA stretching effect of ssDNA is then monitored using single-molecule fluorescence energy transfer (FRET), resulting in decreased FRET efficiency in the stretched ssDNA. By controlling the stretching state of ssDNA, we obtained significantly improved hybridization kinetics (within 1 min) and hybridization efficiency (∼98%) under the target concentration of 500 nM. The bridge DNA sensors demonstrated high sensitivity (1 fM), high specificity (single mismatch mutation discrimination), and high selectivity (suitable for the detection in serum and blood) under the target concentration of 10 nM. Controlling the stretching state of ssDNA shows great potential in biosensors, bioimaging, and biochips applications.

6.
Nat Commun ; 11(1): 2185, 2020 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-32366822

RESUMO

Signal amplification in biological systems is achieved by cooperatively recruiting multiple copies of regulatory biomolecules. Nevertheless, the multiplexing capability of artificial fluorescent amplifiers is limited due to the size limit and lack of modularity. Here, we develop Cayley tree-like fractal DNA frameworks to topologically encode the fluorescence states for multiplexed detection of low-abundance targets. Taking advantage of the self-similar topology of Cayley tree, we use only 16 DNA strands to construct n-node (n = 53) structures of up to 5 megadalton. The high level of degeneracy allows encoding 36 colours with 7 nodes by site-specifically anchoring of distinct fluorophores onto a structure. The fractal topology minimises fluorescence crosstalk and allows quantitative decoding of quantized fluorescence states. We demonstrate a spectrum of rigid-yet-flexible super-multiplex structures for encoded fluorescence detection of single-molecule recognition events and multiplexed discrimination of living cells. Thus, the topological engineering approach enriches the toolbox for high-throughput cell imaging.

7.
ACS Nano ; 14(4): 3747-3754, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32267678

RESUMO

The COVID-19 pandemic is one of those global challenges that transcends territorial, political, ideological, religious, cultural, and certainly academic boundaries. Public health and healthcare workers are at the frontline, working to contain and to mitigate the spread of this disease. Although intervening biological and immunological responses against viral infection may seem far from the physical sciences and engineering that typically work with inanimate objects, there actually is much that can-and should-be done to help in this global crisis. In this Perspective, we convert the basics of infectious respiratory diseases and viruses into physical sciences and engineering intuitions, and through this exercise, we present examples of questions, hypotheses, and research needs identified based on clinicians' experiences. We hope researchers in the physical sciences and engineering will proactively study these challenges, develop new hypotheses, define new research areas, and work with biological researchers, healthcare, and public health professionals to create user-centered solutions and to inform the general public, so that we can better address the many challenges associated with the transmission and spread of infectious respiratory diseases.


Assuntos
Infecções por Coronavirus , Engenharia , Nanotecnologia , Disciplinas das Ciências Naturais , Pandemias , Pneumonia Viral , Betacoronavirus , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/transmissão , Assistência à Saúde , Humanos , Nanotecnologia/tendências , Pneumonia Viral/epidemiologia , Pneumonia Viral/transmissão , Saúde Pública , Editoração
8.
J Am Chem Soc ; 142(19): 8800-8808, 2020 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-32302107

RESUMO

Cells existing in the form of clusters often exhibit distinct physiological functions from their monodispersed forms, which have a close association with tissue and organ development, immunoresponses, and cancer metastasis. Nevertheless, the ability to construct artificial cell clusters as in vitro models for probing and manipulating intercellular communications remains limited. Here we design DNA origami nanostructure (DON)-based biomimetic membrane channels to organize cell origami clusters (COCs) with controlled geometric configuration and cell-cell communications. We demonstrate that programmable patterning of homotypic and heterotypic COCs with different configurations can result in three distinct types of intercellular communications: gap junctions, tunneling nanotubes, and immune/tumor cell interactions. In particular, the organization of T cells and cancer cells with a prescribed ratio and geometry can program in vitro immunoresponses, providing a new route to understanding and engineering cancer immunotherapy.

9.
J Am Chem Soc ; 142(19): 8782-8789, 2020 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-32311267

RESUMO

Ultraviolet (UV) light has long been known to damage nucleic acids. In this work, a DNA origami radiometer has been developed for measuring UV exposure by monitoring the morphological evolution of DNA origami nanostructures. Unlike linear DNA strands that tend to be degraded into small segments upon UV exposure, the structural complexity and interstrand connectivity of DNA origami remarkably alter the pathway of UV-induced DNA damage. A general pathway of expansion, distortion, and final disintegration is observed for DNA origami regardless of their shape and size; however the deformation kinetics is positively correlated with the number of nicks in the nanostructure. This structural continuity-dependent deformation can be translated into a DNA-based radiometer for measuring UV dose in the environment.

10.
Nano Lett ; 20(5): 3521-3527, 2020 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-32223268

RESUMO

Clustering, endocytosis, and intracellular transport of molecules on the cell membrane are critically dependent on the type of cells. However, the membrane-associated redistribution of molecules has not been exploited to realize cell classification for diagnostic purposes. Here, we develop a set of DNA-encoded artificial receptors and ligands to monitor the cell membrane redistribution. In this system, a cholesterol-modified single-stranded DNA strand serves as the receptor localized on the membrane, and a tetrahedral DNA framework (TDF) nanostructure with a complementary overhang serves as the ligand. The DNA-encoded receptor-ligand interaction is highly orthogonal, mimicking the dynamics of natural receptors and ligands on cells. We demonstrate that the dynamics of membrane redistribution can be resolved by the dual-color fluorescent patterns of the receptor-ligand interactions in a single image, which can be exploited to classify cell lines with high fidelity. This DNA-encoded method thus holds great promise for cell typing and diagnosis.

11.
Sci China Life Sci ; 2020 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-32253588

RESUMO

Self-assembled DNA nanostructures have shown remarkable potential in the engineering of biosensing interfaces, which can improve the performance of various biosensors. In particular, by exploiting the structural rigidity and programmability of the framework nucleic acids with high precision, molecular recognition on the electrochemical biosensing interface has been significantly enhanced, leading to the development of highly sensitive and specific biosensors for nucleic acids, small molecules, proteins, and cells. In this review, we summarize recent advances in DNA framework-engineered biosensing interfaces and the application of corresponding electrochemical biosensors.

12.
Artigo em Inglês | MEDLINE | ID: mdl-32267600

RESUMO

Fluorescent copper nanoclusters (CuNCs) have been widely used in chemical sensors, biological imaging, and light-emitting devices. However, individual fluorescent CuNCs have limitations in their capabilities arising from poor photostability and weak emission intensities. As one kind of aggregation-induced emission luminogen (AIEgen), the formation of aggregates with high compactness and good order can efficiently improve the emission intensity, stability, and tunability of CuNCs. Here, DNA nanoribbons, containing multiple specific binding sites, serve as a template for in situ synthesis and assembly of ultrasmall CuNCs (0.6 nm). These CuNC self-assemblies exhibit enhanced luminescence and excellent fluorescence stability because of tight and ordered arrangement through DNA nanoribbons templating. Furthermore, the stable and bright CuNC assemblies are demonstrated in the high-sensitivity detection and intracellular fluorescence imaging of biothiols.

13.
Small ; 16(16): e2000793, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32227454

RESUMO

DNA origami has rapidly emerged as a powerful technique to fabricate user-defined DNA nanostructures. However, the ability to custom-make patterns on DNA origami template is hampered by the heavy workload and high cost of changing staple DNA (up to several hundred strands per set). Here, a scaffold-decorated DNA origami method is developed by prescribing the pattern information to the scaffold DNA. For each pixel of an origami, a designed "pixel strand" (P-strand) is hybridized to the scaffold, strongly preoccupying a specific position and competing with invading staples in a mild origami assembly. To fabricate a new origami pattern, the P-strand set needs to be replaced with a universal staple set. The yield of thus-fabricated DNA origami patterns is comparable to a conventional DNA origami with canonical method. One-pot fabrication of three different nanopatterns in a single test-tube is further demonstrated. Also, dynamic switch of the pattern is shown. This method provides a generic approach and offers large flexibility for scaling up the nanofabrication with DNA origami by kinetically modulating the reaction pathway of the staples with the scaffold DNA, which represents a novel route in the self-assembly of complex biomolecular systems.

14.
Theranostics ; 10(6): 2631-2644, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32194825

RESUMO

Intraoperative image-guided surgery (IGS) has attracted extensive research interests in determination of tumor margins from surrounding normal tissues. Introduction of near infrared (NIR) fluorophores into IGS could significantly improve the in vivo imaging quality thus benefit IGS. Among the reported NIR fluorophores, rare-earth nanoparticles exhibit unparalleled advantages in disease theranostics by taking advantages such as large Stokes shift, sharp emission spectra, and high chemical/photochemical stability. The recent advances in elements doping and morphologies controlling endow the rare-earth nanoparticles with intriguing optical properties, including emission span to NIR-II region and long life-time photoluminescence. Particularly, NIR emissive rare earth nanoparticles hold advantages in reduction of light scattering, photon absorption and autofluorescence, largely improve the performance of nanoparticles in biological and pre-clinical applications. In this review, we systematically compared the benefits of RE nanoparticles with other NIR probes, and summarized the recent advances of NIR emissive RE nanoparticles in bioimaging, photodynamic therapy, drug delivery and NIR fluorescent IGS. The future challenges and promises of NIR emissive RE nanoparticles for IGS were also discussed.

15.
Small ; 16(16): e1904857, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32191376

RESUMO

Effective drug delivery systems that can systematically and selectively transport payloads to disease cells remain a challenge. Here, a targeting ligand-modified DNA origami nanostructure (DON) as an antibody-drug conjugate (ADC)-like carrier for targeted prostate cancer therapy is reported. Specifically, DON of six helical bundles is modified with a ligand 2-[3-(1,3-dicarboxy propyl)-ureido] pentanedioic acid (DUPA) against prostate-specific membrane antigen (PSMA), to serve as the antibody for drug conjugation in ADC. Doxorubicin (Dox) is then loaded to DON through intercalation to dsDNA. This platform features in spatially controllable organization of targeting ligands and high drug loading capacity. With this nanocomposite, selective delivery of Dox to the PSMA+ cancer cell line LNCaP is readily achieved. The consequent therapeutic efficacy is critically dependent on the numbers of targeting ligand assembled on DON. This target-specific and biocompatible drug delivery platform with high maximum tolerated doses shows immense potential for developing novel nanomedicine.

16.
Artigo em Inglês | MEDLINE | ID: mdl-32187784

RESUMO

Molecular recognition in cell biological process is characterized with specific locks-and-keys interactions between ligands and receptors, which are ubiquitously distributed on cell membrane with topological clustering. Few topologically-engineered ligand systems enable the exploration of the binding strength between ligand-receptor topological organization. Herein, we generate topologically controlled ligands by developing a family of tetrahedral DNA frameworks (TDFs), so the multiple ligands are stoichiometrically and topologically arranged. This topological control of multiple ligands changes the nature of the molecular recognition by inducing the receptor clustering, so the binding strength is significantly improved (ca. 10-fold). The precise engineering of topological complexes formed by the TDFs are readily translated into effective binding control for cell patterning and binding strength control of cells for cell sorting. This work paves the way for the development of versatile design of topological ligands.

17.
Nat Commun ; 11(1): 978, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32080196

RESUMO

In order to maintain tissue homeostasis, cells communicate with the outside environment by receiving molecular signals, transmitting them, and responding accordingly with signaling pathways. Thus, one key challenge in engineering molecular signaling systems involves the design and construction of different modules into a rationally integrated system that mimics the cascade of molecular events. Herein, we rationally design a DNA-based artificial molecular signaling system that uses the confined microenvironment of a giant vesicle, derived from a living cell. This system consists of two main components. First, we build an adenosine triphosphate (ATP)-driven DNA nanogatekeeper. Second, we encapsulate a signaling network in the biomimetic vesicle, consisting of distinct modules, able to sequentially initiate a series of downstream reactions playing the roles of reception, transduction and response. Operationally, in the presence of ATP, nanogatekeeper switches from the closed to open state. The open state then triggers the sequential activation of confined downstream signaling modules.


Assuntos
DNA/metabolismo , Transdução de Sinais , Trifosfato de Adenosina/metabolismo , Células Artificiais/química , Materiais Biomiméticos/química , Biomimética/métodos , Homeostase , Nanoestruturas/química , Biologia Sintética/métodos
18.
Nat Commun ; 11(1): 838, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-32047166

RESUMO

Protein-protein interactions are spatially regulated in living cells to realize high reaction efficiency, as seen in naturally existing electron-transfer chains. Nevertheless, arrangement of chemical/biochemical components at the artificial device interfaces does not possess the same level of control. Here we report a tetrahedral DNA framework-enabled bulk enzyme heterojunction (BEH) strategy to program the multi-enzyme catalytic cascade at the interface of electrochemical biosensors. The construction of interpenetrating network of BEH at the millimeter-scale electrode interface brings enzyme pairs within the critical coupling length (CCL) of ~10 nm, which in turn greatly improve the overall catalytic cascade efficiency by ~10-fold. We demonstrate the BEH generality with a range of enzyme pairs for electrochemically detecting clinically relevant molecular targets. As a proof of concept, a BEH-based sarcosine sensor enables single-step detection of the metabolic biomarker of sarcosine with ultrasensitivity, which hold the potential for precision diagnosis of early-stage prostate cancer.


Assuntos
Técnicas Biossensoriais/métodos , DNA/química , Técnicas Eletroquímicas/métodos , Eletrodos , Enzimas Imobilizadas , Técnicas Biossensoriais/instrumentação , Catálise , Técnicas de Química Analítica/métodos , Técnicas Eletroquímicas/instrumentação , Enzimas/química , Desenho de Equipamento , Humanos , Limite de Detecção , Nanopartículas Metálicas , Modelos Teóricos , Nanotecnologia/métodos , Sarcosina
19.
Small ; 16(8): e1907598, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32003943

RESUMO

Lightweight and mechanically strong protein fibers are promising for many technical applications. Despite the widespread investigation of biological fibers based on spider silk and silkworm proteins, it remains a challenge to develop low-cost proteins and convenient spinning technology for the fabrication of robust biological fibers. Since there are plenty of widely available proteins in nature, it is meaningful to investigate the preparation of fibers by the proteins and explore their biomedical applications. Here, a facile microfluidic strategy is developed for the scalable construction of biological fibers via a series of easily accessible spherical and linear proteins including chicken egg, quail egg, goose egg, bovine serum albumin, milk, and collagen. It is found that the crosslinking effect in microfluidic chips and double-drawn treatment after spinning are crucial for the formation of fibers. Thus, high tensile strength and toughness are realized in the fibers, which are comparable or even higher than that of many recombinant spider silks or regenerated silkworm fibers. Moreover, the suturing applications in rat and minipig models are realized by employing the mechanically strong fibers. Therefore, this work opens a new direction for the production of biological fibers from natural sources.

20.
J Am Chem Soc ; 142(6): 2889-2896, 2020 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-31986025

RESUMO

5-Hydroxymethyluracil ( 5hmU ) is found in the genomes of a diverse range of organisms as another kind of 5-hydroxymethylpyrimidine, with the exception of 5-hydroxymethylcytosine ( 5hmC ). The biological function of 5hmU has not been well explored due to lacking both specific 5hmU recognition and single-cell analysis methods. Here we report differentiated visualization of single-cell 5hmU and 5hmC with microfluidic hydrogel encoding (sc 5hmU / 5hmC -microgel). Single cells and their genomic DNA after cell lysis can be encapsulated in individual agarose microgels. The 5hmU sites are then specifically labeled with thiophosphate for the first time, followed by labeling 5hmC with azide glucose. These labeled bases are each encoded into respective DNA barcode primers by chemical cross-linking. In situ amplification is triggered for single-molecule fluorescence visualization of single-cell 5hmU and 5hmC . On the basis of the sc 5hmU / 5hmC -microgel, we reveal cell type-specific molecular signatures of these two bases with remarkable single-cell heterogeneity. Utilizing machine learning algorithms to decode four-dimensional signatures of 5hmU / 5hmC , we visualize the discrimination of nontumorigenic, carcinoma and highly invasive breast cell lines. This strategy provides a new route to analyze and decode single-cell DNA epigenetic modifications.

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