Enzyme-Programmed Self-Assembly of Nanoparticles.

Nanoparticles are a hot topic in the field of nanomaterial research due to their excellent physical and chemical properties. In recent years, DNA-directed nanoparticle self-assembly technology has been widely applied to the development of numerous complex nanoparticle superstructures. Due to the inherent stability and surface electric repulsion of nanoparticles, it is difficult to make nanoparticle superstructures respond to molecular signals in the external environment. In fact, enzyme-programmed molecular systems are developed to allow diverse functions, including logical operations, signal amplification, and dynamic assembly control. Therefore, combining enzyme-controlled DNA systems may endow nanoparticle assembly systems with more flexibility in program design, allowing them to respond to a variety of external signals. In this review, we summarize the basic principles of enzyme-controlled DNA/nanoparticle self-assembly and introduce its applications in heavy metal detection, gene expression, proteins inside living cells, cancer cell therapy, and drug delivery. With the continuous development of new nanoparticle materials and the increasing functionality of enzyme DNA circuits, enzyme-directed DNA/nanoparticle self-assembled probe technology is expected to see significant future development.

Programmable Primer Switching for Regulating Enzymatic DNA Circuits.

Developing DNA strand displacement reactions (SDRs) offers crucial technical support for regulating artificial nucleic acid circuits and networks. More recently, enzymatic SDR-based DNA circuits have gained significant attention because of their modular design, high orthogonality signaling, and extremely fast reaction rates. Typical enzymatic SDRs are regulated by relatively long primers (20-30 nucleotides) that hybridize to form stable double-stranded structures, facilitating enzyme-initiated events. Implementing more flexible primer-based enzymatic SDR regulations remains challenging due to the lack of convenient and simple primer control mechanism, which consequently limits the development of enzymatic DNA circuits. In this study, we propose an approach, termed primer switching regulation, that implements programmable and flexible regulations of enzymatic circuits by introducing switchable wires into the enzymatic circuits. We applied this method to generate diverse enzymatic DNA circuits, including cascading, fan-in/fan-out, dual-rail, feed-forward, and feedback functions. Through this method, complex circuit functions can be implemented by just introducing additional switching wires without reconstructing the basic circuit frameworks. The method is experimentally demonstrated to provide flexible and programmable regulations to control enzymatic DNA circuits and has future applications in DNA computing, biosensing, and DNA storage.

Catalytic Gold Nanoparticle Assembly Programmed by DNAzyme Circuits.

Assembled gold nanoparticle (AuNP) superstructures can generate unique physicochemical characteristics and be used in various applications, thus becoming an attractive research field. Recently, several DNA-assisted gold nanoparticle assembly methods have been rigorously developed that typically require a non-catalytic equimolar molecular assembly to guarantee the designed assembly. Although efficient and accurate, exploring such non-catalytic nanoparticle assemblies in the complex cellular milieu under low trigger concentrations remains challenging. Therefore, developing a catalytic method that facilitates gold nanoparticle assemblies with relatively low DNA trigger concentrations is desirable. In this report, a catalytic method to program gold nanoparticle assemblies by DNAzyme circuits is presented, where only a small number of DNA triggers are able to induce the production of a large number of the desired nanoparticle assemblies. The feasibility of using logic DNAzyme circuits to control catalytic nanoparticle assemblies is experimentally verified. Additionally, catalytic AuNP assembly systems are established with cascading and feedback functions. The work provides an alternative research direction to enrich the tool library of nanoparticle assembly and their application in biosensing and nanomedicine.

Programming conformational cooperativity to regulate allosteric protein-oligonucleotide signal transduction.

Conformational cooperativity is a universal molecular effect mechanism and plays a critical role in signaling pathways. However, it remains a challenge to develop artificial molecular networks regulated by conformational cooperativity, due to the difficulties in programming and controlling multiple structural interactions. Herein, we develop a cooperative strategy by programming multiple conformational signals, rather than chemical signals, to regulate protein-oligonucleotide signal transduction, taking advantage of the programmability of allosteric DNA constructs. We generate a cooperative regulation mechanism, by which increasing the loop lengths at two different structural modules induced the opposite effects manifesting as down- and up-regulation. We implement allosteric logic operations by using two different proteins. Further, in cell culture we demonstrate the feasibility of this strategy to cooperatively regulate gene expression of PLK1 to inhibit tumor cell proliferation, responding to orthogonal protein-signal stimulation. This programmable conformational cooperativity paradigm has potential applications in the related fields.

Development of a neuron model based on DNAzyme regulation.

Neural networks based on DNA molecular circuits play an important role in molecular information processing and artificial intelligence systems. In fact, some DNA molecular systems can become dynamic units with the assistance of DNAzymes. The complex DNA circuits can spontaneously induce corresponding feedback behaviors when their inputs changed. However, most of the reported DNA neural networks have been implemented by the toehold-mediated strand displacement (TMSD) method. Therefore, it was important to develop a method to build a neural network utilizing the TMSD mechanism and adding a mechanism to account for modulation by DNAzymes. In this study, we designed a model of a DNA neuron controlled by DNAzymes. We proposed an approach based on the DNAzyme modulation of neuronal function, combing two reaction mechanisms: DNAzyme digestion and TMSD. Using the DNAzyme adjustment, each component simulating the characteristics of neurons was constructed. By altering the input and weight of the neuron model, we verified the correctness of the computational function of the neurons. Furthermore, in order to verify the application potential of the neurons in specific functions, a voting machine was successfully implemented. The proposed neuron model regulated by DNAzymes was simple to construct and possesses strong scalability, having great potential for use in the construction of large neural networks.

Entropy-driven DNA logic circuits regulated by DNAzyme.

The catalytic DNA circuits play a critical role in engineered biological systems and molecular information processing. Actually, some of the natural or synthetic DNA circuits were triggered by covalent modifications, where conformational changes were induced to facilitate complex DNA engineering functions and signal transmissions. However, most of the reported artificial catalytic DNA circuits were regulated by the toehold-mediated reaction. Therefore, it is significant to propose a strategy to regulate the catalytic DNA circuit not only by the toehold-mediated mechanism, but also by involving the conformational changes induced by the covalent modification. In this study, we developed the catalytic DNA logic circuits regulated by DNAzyme. Here, a regulation strategy based on the covalent modification was proposed to control the DNA circuit, combing two reaction mechanisms: DNAzyme digestion and entropy-driven strand displacement. The DNAzyme and DNA catalyst can participate into the reactions alternatively, thus realizing the cascading catalytic circuits. Using the DNAzyme regulation, a series of logic gates (YES, OR and AND) were constructed. In addition, a two-layer cascading circuit and a feedback self-catalysis circuit were also established. The proposed DNAzyme-regulated strategy shows great potentials as a reliable and feasible method for constructing more complex catalytic DNA circuits.

Programmable Regulation of DNA Conjugation to Gold Nanoparticles via Strand Displacement.

Methods for conjugating DNA to gold nanoparticles (AuNPs) have recently attracted considerable attention. The ability to control such conjugation in a programmable way is of great interest. Here, we have developed a logic-based method for manipulating the conjugation of thiolated DNA species to AuNPs via cascading DNA strand displacement. Using this method, several logic-based operation systems are established and up to three kinds of DNA signals are introduced at the same time. In addition, a more sensitive catalytic logic-based operation is also achieved based on an entropy-driven process. In the experiment, all of the DNA/AuNPs conjugation results are verified by agrose gel. This strategy promises great potential for automatically conjugating DNA stands onto label-free gold nanoparticles and can be extended to constructing DNA/nanoparticle devices for applications in diagnostics, biosensing, and molecular robotics.