Caging-Decaging Strategies to Realize Spatiotemporal Control of DNAzyme Activity for Biosensing and Bioimaging

DNAzymes with RNA-cleaving activity have been widely used as biosensing and bioimaging tools for detection of metal ions.Despite the achievements,DNAzyme-based biosensors sometime suffer from false positive signals and unexpected off-target turn-on in biological environments,which are likely due to the unstable nature of the RNA site.Ways to control DNAzyme activity in order to improve the sensing performance remain a significant challenge.To meet the challenge,there is growing interest to develop synthetic strategies that can cage native DNAzyme under undesired conditions and reactivate it in target environment in order to function in a controlled manner.A variety of caging-decaging strategies have been developed to realize spatiotemporal control of the DNAzyme activity,improving its specificity and sensitivity as well as extending its application regimes.In this review,we focus on strategies to regulate the catalytic activity of DNAzyme,highlight the nucleic acid modification chemistries,and summarize three strategies to cage DNAzyme functions.Examples of using caged DNAzyme for bio-applications have also been reviewed in detail.Finally,we provide our perspectives on the potential challenges and opportunities of this emerging research topic that could advance the DNAzyme field.

Intracavity spatiotemporal metasurfaces

Optical metasurfaces are endowed with unparallel flexibility to manipulate the light field with a subwavelength spatial resolution.Coupling metasurfaces to materials with strong optical nonlinearity may allow ultrafast spatiotemporal light field modulation.However,most metasurfaces demonstrated thus far are linear devices.Here,we experimentally demonstrate simultaneous spatiotemporal laser mode control using a single-layer plasmonic metasurface strongly coupled to an epsilon-near-zero(ENZ)material within a fiber laser cavity.While the geometric phase of the metasurface is utilized to convert the laser’s transverse mode from a Gaussian beam to a vortex beam carrying orbital angular momentum,the giant nonlinear saturable absorption of the ENZ material enables pulsed laser generation via the Q-switching process.The direct integration of a spatiotemporal metasurface in a laser cavity may pave the way for the development of miniaturized laser sources with tailored spatial and temporal profiles,which can be useful for numerous applications,such as superresolution imaging,high-density optical storage,and three-dimensional laser lithography.

Design and mathematical analysis of activating transcriptional amplifiers that enable modular temporal control in synthetic juxtacrine circuits

The ability to control mammalian cells such that they self-organize or enact therapeutic effects as desired has incredible implications.Not only would it further our understanding of native processes such as development and the immune response,but it would also have powerful applications in medical fields such as regenerative medicine and immunotherapy.This control is typically obtained by synthetic circuits that use synthetic receptors,but control remains incomplete.The synthetic juxtacrine receptors(SJRs)are widely used as they are fully modular and enable spatial control,but they have limited gene expression amplification and temporal control.As these are integral facets to cell control,I therefore designed transcription factor based amplifiers that amplify gene expression and enable unidirectional temporal control by prolonging duration of target gene expression.Using a validated in silico framework for SJR signaling,I combined these amplifiers with SJRs and show that these SJR amplifier circuits can direct spatiotemporal patterning and improve the quality of self-organization.I then show that these circuits can improve chimeric antigen receptor(CAR)T cell tumor killing against various heterogenous antigen expression tumors.These amplifiers are flexible tools that improve control over SJR based circuits with both basic and therapeutic applications.

Conditional gene expression in invertebrate animal models

A mechanistic understanding of biology requires appreciating spatiotemporal aspects of gene expression and its functional implications.Conditional expression allows for (ir)reversible switching of genes on or off,with the potential of spatial and/or temporal control.This provides a valuable complement to the more often used constitutive gene (in)activation through mutagenesis,providing tools to answer a wider array of research questions across biological disciplines.Spatial and/or temporal control are granted primarily by(combinations of) specific promoters,temperature regimens,compound addition,or illumination.The use of such genetic tool kits is particularly widespread in invertebrate animal models because they can be applied to study biological processes in short time frames and on large scales,using organisms amenable to easy genetic manipulation.Recent years witnessed an exciting expansion and optimization of such tools,of which we provide a comprehensive overview and discussion regarding their use in invertebrates.The mechanism,applicability,benefits,and drawbacks of each of the systems,as well as further developments to be expected in the foreseeable future,are highlighted.

Growth factor delivery using extracellular matrix-mimicking substrates for musculoskeletal tissue engineering and repair

Therapeutic approaches for musculoskeletal tissue regeneration commonly employ growth factors(GFs)to influence neighboring cells and promote migration,proliferation,or differentiation.Despite promising results in preclinical models,the use of inductive biomacromolecules has achieved limited success in translation to the clinic.The field has yet to sufficiently overcome substantial hurdles such as poor spatiotemporal control and supraphysiological dosages,which commonly result in detrimental side effects.Physiological presentation and retention of biomacromolecules is regulated by the extracellular matrix(ECM),which acts as a reservoir for GFs via electrostatic interactions.Advances in the manipulation of extracellular proteins,decellularized tissues,and synthetic ECM-mimetic applications across a range of biomaterials have increased the ability to direct the presentation of GFs.Successful application of biomaterial technologies utilizing ECM mimetics increases tissue regeneration without the reliance on supraphysiological doses of inductive biomacromolecules.This review describes recent strategies to manage GF presentation using ECM-mimetic substrates for the regeneration of bone,cartilage,and muscle.

Physical stimuli-responsive cell-free protein synthesis

Cell-free protein synthesis has been developed as a critical platform in synthetic biology.Unlike the cell-based synthesis system,cell-free system activates transcriptional and translational mechanisms in vitro,and can control protein synthesis by artificially adding components or chemicals.However,the control method puts forward higher requirements in terms of accurate and non-toxic control,which cannot be achieved by chemical substances.For cell-free system,physical signal is a kind of ideal spatiotemporal control approach to replace chemical substances,realizing high accuracy with little side effect.Here we review the methods of using physical signals to control gene expression in cell-free systems,including studies based on light,temperature,electric field,and magnetic force.The transfer of these switches into cell-free system further expands the flexibility and controllability of the system,thus further expanding the application capability of cell-free systems.Finally,existing problems such as signal source and signal transmission are discussed,and future applications in pharmaceutical production,delivery and industrial production are further looked into.