Catalytic-assembly of programmable atom equivalents.

Escape from metastable states in self-assembly of colloids is an intractable problem. Unlike the commonly adopted approach of thermal annealing, the recently developed enthalpy-mediated strategy provided a different option to address this dilemma in a dynamically controllable manner at room temperature. However, it required a complex catalytic-assembly DNA strand-displacement circuitry to mediate interaction between multiple components. In this work, we present a simple but effective way to achieve catalytic-assembly of DNA-functionalized colloidal nanoparticles, i.e., programmable atom equivalents, in a far-from-equilibrium system. A removable molecule named "catassembler" that acts as a catalyst was employed to rectify imperfect linkages and help the system escape from metastability without affecting the assembled framework. Notably, catalytic efficiency of the catassembler can be effectively improved by changing the seesaw catassembler in toehold length design or numbers of the repeat units. Leveraging this tractable catalytic-assembly approach, different ordered architectures were easily produced by directly mixing all reactants, as in chemical reactions. By switching bonding identities, solid-solid phase transformations between different colloidal crystals were achieved. This work opens up an avenue for programming colloid assembly in a far-from-equilibrium system.

Efficient precise integration of large DNA sequences with 3'-overhang dsDNA donors using CRISPR/Cas9.

CRISPR/Cas9 genome-editing tools have tremendously boosted our capability of manipulating the eukaryotic genomes in biomedical research and innovative biotechnologies. However, the current approaches that allow precise integration of gene-sized large DNA fragments generally suffer from low efficiency and high cost. Herein, we developed a versatile and efficient approach, termed LOCK (Long dsDNA with 3'-Overhangs mediated CRISPR Knock-in), by utilizing specially designed 3'-overhang double-stranded DNA (odsDNA) donors harboring 50-nt homology arm. The length of the 3'-overhangs of odsDNA is specified by the five consecutive phosphorothioate modifications. Compared with existing methods, LOCK allows highly efficient targeted insertion of kilobase-sized DNA fragments into the mammalian genomes with low cost and low off-target effects, yielding >fivefold higher knock-in frequencies than conventional homologous recombination-based approaches. This newly designed LOCK approach based on homology-directed repair is a powerful tool suitable for gene-sized fragment integration that is urgently needed for genetic engineering, gene therapies, and synthetic biology.

pH-Controlled Resettable Modular DNA Strand-Displacement Circuits.

Sophisticated dynamic molecular systems with diverse functions have been fabricated by using the fundamental tool of toehold-mediated strand displacement (TMSD) in the field of dynamic DNA nanotechnology. However, simple approaches to reset these TMSD-based dynamic systems are lacking due to the difficulty in creating kinetically favored pathways to implement the backward resetting reactions. Here, we develop a facile proton-driven strategy to achieve complete resetting of a modular DNA circuit by integrating a pH-responsive intermolecular CG-C+ triplex DNA and an i-motif DNA into the conventional DNA substrate. The pH-programmed strategy allows modular DNA components to specifically associate/dissociate to promote the forward/backward TMSD reactions, thereby enabling the modular DNA circuit to be repeatedly operated at a constant temperature without generating any DNA waste products. Leveraging this tractable approach, we further constructed two resettable DNA logic gates used for logical computation and two resettable catalytic DNA systems with good performance in signal transduction and amplification.

Programming of Supercrystals Using Replicable DNA-Functionalized Colloids.

The development of self-replicating systems is of great importance in research on the origin of life. As the most iconic molecules, nucleic acids have provided prominent examples of the fabrication of self-replicating artificial nanostructures. However, it is still challenging to construct sophisticated synthetic systems that can create large-scale or three-dimensionally ordered nanomaterials using self-replicating nanostructures. By integrating a template system containing DNA-functionalized colloidal seeds with a simplified DNA strand-displacement circuit programmed subsystem to produce DNA-functionalized colloidal copies, we developed a facile enthalpy-mediated strategy to control the replication and catalytic assembly of DNA-functionalized colloids in a time-dependent manner. The replication efficiency and crystal quality of the resulting superlattice structures can be effectively increased by regulating the molar ratio of the template to the copy colloids. By constructing binary systems from two types of gold nanoparticles (or proteins), superlattice structures with different crystal symmetries can be obtained through the replication and catalytic assembly processes. This programmable enthalpy-mediated approach was easily leveraged to achieve the phase transformation and catalytic amplification of colloidal crystals starting from different initial template crystals. This work offers a potential way to construct self-replicating artificial systems that exhibit complicated phase behaviors and can produce large-scale superlattice nanomaterials.

Catalytic assembly of symmetric diblock copolymers in a thin film: a dissipative particle dynamics simulation study.

Obtaining a thin block copolymer film with a perfect structure by self-assembly is difficult because the system is, in general, trapped in a metastable state. We used dissipative particle dynamics (DPD) to investigate the self-assembly of AB symmetric diblock copolymers in a thin film. We discovered that addition of a small molecule (molecule C) as the third composition could help the system evade the metastable state. Therefore, imperfect structures could be corrected, and ordered structures formed. Analogous to the performance of a catalyst in catalytic chemistry, molecule C could promote assembly into an ordered structure, but was less involved within the polymer phase. Moreover, simulation results showed that the content of molecule C and its repulsive interactions with blocks A and B were quite important for promoting assembly into ordered structures effectively.

Programming colloidal bonding using DNA strand-displacement circuitry.

As a strategy for regulating entropy, thermal annealing is a commonly adopted approach for controlling dynamic pathways in colloid assembly. By coupling DNA strand-displacement circuits with DNA-functionalized colloid assembly, we developed an enthalpy-mediated strategy for achieving the same goal while working at a constant temperature. Using this tractable approach allows colloidal bonding to be programmed for synchronization with colloid assembly, thereby realizing the optimal programmability of DNA-functionalized colloids. We applied this strategy to conditionally activate colloid assembly and dynamically switch colloid identities by reconfiguring DNA molecular architectures, thereby achieving orderly structural transformations; leveraging the advantage of room-temperature assembly, we used this method to prepare a lattice of temperature-sensitive proteins and gold nanoparticles. This approach bridges two subfields: dynamic DNA nanotechnology and DNA-functionalized colloid programming.

Programmable Transcriptional Modulation with a Structured RNA-Mediated CRISPR-dCas9 Complex.

Multi-module dCas9 engineering systems have been developed for controllable transcriptional manipulation such as chemical- or light-induced systems. However, there is still a need for a separate module that can be used for internal control over the CRISPR-dCas9 system. Here, we describe a multi-module CRISPR-dCas9 system in which a separate structured RNA was applied as a programmable component that could control dCas9-based gene regulation and achieved a higher activation efficiency than dCas9-VPR that is traditionally used. By introducing a microRNA sensor, we generated a dCas9-based transcriptional regulation platform that responded to endogenous microRNAs and allowed controllable activation of endogenous genes. Moreover, we applied the platform to selectively identify HCT116 cells in a cell mixture. This work provides a flexible platform for efficient and controllable gene regulation based on CRISPR-dCas9.

Identifying and Differentiating Topological G-Quadruplex Structures with DNA-Encoded Plasmonic Gold Nanoparticles.

DNA G-quadruplexes (G4s) have been identified as critical elements in modulating genomic functions and many other biological processes. Their functions are highly dependent on the primary nucleotides and secondary folding structures. Therefore, to understand their functions, methods to identify and differentiate structures of G4 with speed and accuracy are required but limited. In this report, we have applied a synthetic G4 DNA-encoded nanoparticle approach to identify and differentiate G4 DNA molecules with different topologies and nucleotide residues. We found that the resulting plasmonic properties of the gold nanoparticles, monitored by UV/Vis spectroscopy, are quite sensitive to different G4 structures, including stacking layers, loop sequences, capping bases on G4s, and topological structures. Through these systematic investigations, we demonstrate that this G4-encoded gold nanoparticle approach can be used to profile the G4 structures and distinguish G4s from human telomeres. Such a method may have wide applications in G4 research.

A Scalable "Junction Substrate" to Engineer Robust DNA Circuits.

Versatile building blocks are essential for building complex and scaled-up DNA circuits. In this study, we propose a conceptually new scalable architecture called a "junction substrate" (J-substrate) that is linked by prepurified double-stranded DNA molecules. As a proof-of-concept, this novel type of substrate has been utilized to build multi-input DNA circuits, offering several advantages over the conventional substrate (referred to as a "linear substrate", L-substrate). First, the J-substrate does not require long DNA strands, thus avoiding significant synthetic errors and costs. Second, the traditional PAGE purification method is technically facilitated to obtain high-purity substrates, whereby the initial leakage is effectively eliminated. Third, the asymptotic leakage is eliminated by introducing the "junction". Finally, circuits with the optimized J-substrate architecture exhibit fast kinetics. We believe that the proposed architecture constitutes a sophisticated chassis for constructing complex circuits.

Optimizing the Toehold Strategy of On-Chip Nucleic Acid Hybridization Probe for the Discrimination of Single Nucleotide Polymorphism.

The synthetic DNA hybridization probe has proved its importance in biology and biotechnology. In this study, taking advantage of a novel analytical technique called dual polarization interferometry (DPI), the influence of the toehold strategy of on-chip DNA hybridization probe on the discrimination of single nucleotide polymorphism (SNP) was investigated. Through adjusting the toehold length, the toehold strategies of on-chip toehold exchange probe were thoroughly optimized. For the "6/5" probe, an optimal discrimination factor of 78% against the spurious target was achieved. Moreover, the ability of the on-chip probe in SNP discrimination was significantly enhanced compared to its pure solution counterpart. This simple and rapid detection method for SNP discrimination based on the on-chip toehold exchange probe will show great potential in disease diagnosis.

Integrating DNA-strand-displacement circuitry with self-assembly of spherical nucleic acids.

Programmable and algorithmic behaviors of DNA molecules allow one to control the structures of DNA-assembled materials with nanometer precision and to construct complex networks with digital and analog behaviors. Here we developed a way of integrating a DNA-strand-displacement circuit with self-assembly of spherical nucleic acids, wherein a single DNA strand was used to initiate and catalyze the operation of upstream circuits to release a single strand that subsequently triggers self-assembly of spherical nucleic acids in downstream circuits, realizing a programmable kinetic control of self-assembly of spherical nucleic acids. Through utilizing this method, single-nucleotide polymorphisms or indels occurring at different positions of a sequence of oligonucleotide were unambiguously discriminated. We provide here a sophisticated way of combining the DNA-strand-displacement-based characteristic of DNA with the distinct assembly properties of inorganic nanoparticles, which may find broad potential applications in the fabrication of a wide range of complex multicomponent devices and architectures.

DNA Polymer Brush Patterning through Photocontrollable Surface-Initiated DNA Hybridization Chain Reaction.

The fabrication of DNA polymer brushes with spatial resolution onto a solid surface is a crucial step for biochip research and related applications, cell-free gene expression study, and even artificial cell fabrication. Here, for the first time, a DNA polymer brush patterning method is reported based on the photoactivation of an ortho-nitrobenzyl linker-embedded DNA hairpin structure and a subsequent surface-initiated DNA hybridization chain reaction (HCR). Inert DNA hairpins are exposed to ultraviolet light irradiation to generate DNA duplexes with two active sticky ends (toeholds) in a programmable manner. These activated DNA duplexes can initiate DNA HCR to generate multifunctional patterned DNA polymer brushes with complex geometrical shapes. Different multifunctional DNA polymer brush patterns can be fabricated on certain areas of the same solid surface using this method. Moreover, the patterned DNA brush surface can be used to capture target molecules in a desired manner.

What Controls the "Off/On Switch" in the Toehold-Mediated Strand Displacement Reaction on DNA Conjugated Gold Nanoparticles?

In DNA dynamic nanotechnology, a toehold-mediated DNA strand-displacement reaction has demonstrated its capability in building complex autonomous system. In most cases, the reaction is performed in pure DNA solution that is essentially a one-phase system. In the present work, we systematically investigated the reaction in a heterogeneous media, in which the strand that implements a displacing action is conjugated on gold nanoparticles. By monitoring the kinetics of spherical nucleic acid (SNA) assembly driven by toehold-mediated strand displacement reaction, we observed significant differences, i.e., the abrupt jump in behavior of an "off/on switch", in the reaction rate when the invading toehold was extended to eight bases from seven bases. These phenomena are attributed to the effect of steric hindrance arising from the high density of invading strand conjugated to AuNPs. Based on these studies, an INHIBIT logic gate presenting good selectivity was developed.

The combination of gold nanorods and nanoparticles with DNA nanodevices for logic gates construction.

In this work, two DNA nanodevices were constructed utilizing a DNA strand displacement reaction. With the assistance of gold nanoparticles (AuNPs) and gold nanorods (AuNRs), the autonomous reactions can be reflected from the aggregation states of nanoparticles. By sequence design and the two non-overlapping double hump-like UV-vis spectral peaks of AuNPs and AuNRs, two logic gates with multiple inputs and outputs were successfully run with expected outcomes. This method not only shows how to achieve computing with multiple logic calculations but also has great potential for multiple targets detection.

Unexploited Performance of NLS in the dCas9-VPR-Mediated Transcriptional Activation.

Nuclear localization signal (NLS) is a short peptide guiding the nuclear transport process, recognized as playing an important role in constructing clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) activators. Here, we investigate the effect of the position and number of the NLS on transcriptional activation based on the dCas9-VPR activator. Our results not only demonstrate that the position of the SV40 NLS could have different degrees of influence on activation efficiency but also, surprisingly, we find that the SV40 NLS plays a detrimental role. Complete deletion of the NLS from the system could increase the transcriptional activation efficiency by 2 to 4 times compared with the original dCas9-VPR. This finding is also supported by some typical first- and third-generation activators. Our work should be beneficial to the design of the NLS-based system.

A facile biosynthesis strategy of plasmid DNA-derived nanowires for readable microRNA logic operations.

Multiple microRNA (miRNA) logical assays have attracted wide attention recently, which can be applied to mimic and reveal cellular events at the molecular level. However, it remains challenging to develop labeling- and amplification-free approaches to perform logical functions with low levels of miRNA molecules. Herein, we proposed a strategy for miRNA logic operations using plasmid DNA-derived nanowires produced from a facile biosynthesis method. First, let-7d was chosen as the model target of the plasmid DNA-derived nanowire strategy, which showed good selectivity and a response sensitivity of as low as the femtomolar level. The operations of the miRNA logic gates proved the programmability of the constructed plasmid DNA-derived nanowire system for two inputs (let-7d and miR-21). Finally, three pairs of DNA nanowires were combined together to demonstrate the availability of this strategy in parallel multiple miRNAs assays. In this strategy, readout signals can be directly obtained from agarose gel without extra chemical labeling or amplification procedures. Considering the excellent performance of the logic gates with low levels of inputs, our plasmid DNA-derived nanowire strategy could provide a facile method to promote simultaneous multiple miRNA assays for the benefit of diagnosis and could be applied for the assembly of complex DNA nanostructures.

A rational design of a cascaded DNA circuit for nanoparticle assembly and its application in the discrimination of single-base changes.

In the field of dynamic DNA nanotechnology, a designable DNA assembly circuit based on the toehold-mediated strand displacement reaction has demonstrated its ability to program the self-assembly of nanoparticles. However, the laborious work for the modification of nanoparticles with oligonucleotides, the long assembly time, and the circuit leakage prevent its further and scalable applications. To this end, cascaded circuits composed of two recycling circles are rationally designed in this study. Through the pre-initiation of the autonomous reaction, nanoparticles as sensing elements and no additionally exposed bases on the substrate hybridized with fuel strand, the real assembly time and signal leakage for diagnostic application can be effectively reduced and eliminated, thus offering a promising methodology for future point-of-care testing.

Cadherin-dependent adhesion modulated 3D cell-assembly.

The emergence of synthetic biology has opened new avenues in constructing cell-assembly biosystems with specific gene expression and function. The phenomena of cell spreading and detachment during tissue development and cancer metastasis are caused by surface tension, which in turn results from differences in cell-cell adhesion mediated by the dimerization of cadherin expressed on the cell surface. In this study, E- and P-cadherin plasmids were first constructed based on the differential adhesion hypothesis, then they were electroporated into K562 cells and HEK293T cells, respectively, to explore the process of cell migration and assembly regulated by cadherins. Using this approach, some special 3D cell functional components with a phase separation structure were fabricated successfully. Our work will be of potential application in the construction of self-assembling synthetic tissues and organoids.

Nanodelivery of nucleic acids.

There is growing need for a safe, efficient, specific and non-pathogenic means for delivery of gene therapy materials. Nanomaterials for nucleic acid delivery offer an unprecedented opportunity to overcome these drawbacks; owing to their tunability with diverse physico-chemical properties, they can readily be functionalized with any type of biomolecules/moieties for selective targeting. Nucleic acid therapeutics such as antisense DNA, mRNA, small interfering RNA (siRNA) or microRNA (miRNA) have been widely explored to modulate DNA or RNA expression Strikingly, gene therapies combined with nanoscale delivery systems have broadened the therapeutic and biomedical applications of these molecules, such as bioanalysis, gene silencing, protein replacement and vaccines. Here, we overview how to design smart nucleic acid delivery methods, which provide functionality and efficacy in the layout of molecular diagnostics and therapeutic systems. It is crucial to outline some of the general design considerations of nucleic acid delivery nanoparticles, their extraordinary properties and the structure-function relationships of these nanomaterials with biological systems and diseased cells and tissues.

Minimizing Leakage in Stacked Strand Exchange Amplification Circuits.

Signal amplification is ubiquitous in biology and engineering. Protein enzymes, such as DNA polymerases, can routinely achieve >106-fold signal increase, making them powerful tools for signal enhancement. Considerable signal amplification can also be achieved using nonenzymatic, cascaded nucleic acid strand exchange reactions. However, the practical application of such kinetically trapped circuits has so far proven difficult due to uncatalyzed leakage of the cascade. We now demonstrate that strategically positioned mismatches between circuit components can reduce unprogrammed hybridization reactions and therefore greatly diminish leakage. In consequence, we were able to synthesize a three-layer catalytic hairpin assembly cascade that could operate in a single tube and that yielded 3.7 × 104-fold signal amplification in only 4 h, a greatly improved performance relative to previous cascades. This advance should facilitate the implementation of nonenzymatic signal amplification in molecular diagnostics, as well as inform the design of a wide variety of increasingly intricate nucleic acid computation circuits.