Bioimaging Based on Nucleic Acid Nanostructures

Nucleic acid nanostructures with structural programmability, spatial addressability and excellent biocompatibility have drawn much attention in various biomedical applications, such as bioimaging, biosensing and drug delivery. In this review, we summarize the recent research progress in the field of bioimaging based on nucleic acid nanostructures with different imaging models, including fluorescent imaging(FI), magnetic resonance imaging(MRI), photoacoustic imaging(PAI) and positron emission tomography/computed tomography(PET/CT) imaging. We also discuss the remaining challenges and further opportunities involved in the bioimaging research based on nucleic acid nanostructures.     

Recent development on peptide-based probes for multifunctional biomedical imaging

Peptide-based probes play prominent roles in biomedical research due to their promising properties such as high biocompatibility, fast excretion, favorable pharmacokinetics as well as easy and robust preparation. Considering the translation of imaging probes into clinical applications, peptide-based probes remain to be the most desirable and optimal candidates.     

64Cu radiolabeled nanomaterials for positron emission tomography (PET) imaging

The prevalence of positron emission tomography (PET) imaging has advanced biomedical applications for its ultrahigh sensitivity, deep tissue penetration and quantitative visualization of diseases in vivo. ⁶⁴Cu with ideal half-life and decay characteristics has been designed as radioactive probes for disease diagnosis. The currently reported ⁶⁴Cu-labeled nanomaterials have the advantages of long circulation time in serum, good biocompatibility and mature preparation methods, and have been used in vivo PET imaging, biodistribution and pharmacokinetic monitoring, and imaging guided therapy. At the same time, suitable carrier characteristics and radiolabeling strategies are particularly important in the ⁶⁴Cu PET imaging process. In this review, we summarize different imaging probe designs and ⁶⁴Cu radiolabeling strategies, as well as their eventual applications in biomedicine. The potential challenges and prospects of ⁶⁴Cu labeled nanomaterials are also described, which provides broad prospects for radiolabeling strategies and further applications.     

基于DNA纳米结构的细胞间相互作用的调控

细胞通过化学信号、 电子交换和直接接触等方式交换彼此之间的物质和信息, 以调节生命体的生长发育. 因此, 细胞间的相互作用研究与调控在细胞功能的机制研究和疾病的诊断及治疗等领域具有非常重要的意义. DNA纳米结构具有易合成、 易修饰、 可编程性设计及生物安全性高等优点, 有望实现操作简单、 精确可调、 智能响应的细胞间相互作用调控, 受到了广泛关注. 本文综述了寡核苷酸链杂交、 受体-配体结合和核酸适体靶向识别等基于DNA纳米结构的细胞组装策略, 总结了pH调控、 金属离子调控和DNA链激活等细胞间相互作用的调控手段, 并重点介绍了其在细胞间作用力的测量和成像、 体外组织模型的构建、 细胞间的通讯交流和细胞免疫治疗等领域的应用. 最后对该领域进行了总结和展望, 希望为相关研究提供有益参考.     

Construction of Smart Stimuli-Responsive DNA Nanostructures for Biomedical Applications

DNA nanostructures have recently attracted increasing interest in biological and biomedical applications by virtue of their unique properties, such as structural programmability, multi-functionality, stimuli-responsive behaviors, and excellent biocompatibility. In particular, the intelligent responsiveness of smart DNA nanostructures to specific stimuli has facilitated their extensive development in the field of high-performance biosensing and controllable drug delivery. This minireview begins with different self-assembly strategies for the construction of various DNA nanostructures, followed by the introduction of a variety of stimuli-responsive functional DNA nanostructures for assembling metastable soft materials and for facilitating amplified biosensing. The recent achievements of smart DNA nanostructures for controllable drug delivery are highlighted. Finally, the current challenges and possible developments of this promising research are discussed in the fields of intelligent nanomedicine.     

DNA银纳米簇在功能核酸荧光生物传感器中的应用

DNA银纳米簇(DNA-AgNCs)是以DNA为模板, 通过碱基杂环上的N原子与Ag⁺结合, 用NaBH₄将Ag⁺还原得到的具有荧光性质的新兴纳米探针. 由于DNA-AgNCs具有合成方法简单、 生物相容性好和荧光发射波长可调等优点, 使其在分析检测等领域具有广泛的应用. 本文对DNA-AgNCs的合成和荧光性质两个方面进行了综述, 分类总结了以DNA-AgNCs为无标记荧光探针在功能核酸荧光生物传感器方面的应用, 对其不足与应用潜力进行展望, 以期为未来的研究与应用提供借鉴.     

Molecularly near-infrared fluorescent theranostics for in vivo tracking tumor-specific chemotherapy

Molecularly near-infrared(NIR) theranostics, combining in vivo sensing and tumor-specific therapeutic capability within one molecular system, have received considerable attention in recent years. Compared with the visible fluorescence imaging, NIR imaging(emission wavelength at 650–900 nm) possesses unique advantages including the minimum photodamage to biological samples, deep penetration, and low interference from auto-fluorescence. In over past decades, there has been an explosive development in the design of molecular imaging contrasts and imaging-guided therapeutics. In this review, we have sumarried the strategies of the NIR theranostics for imaging and tumor-specific chemotherapy applications in living systems. It is noted that the molecularly NIR theranostic design strategy could address current challenges of real-time in vivo sense-and-release for the intelligent biosensing and personalized treatment.     

A Semisynthetic Bioluminescence Sensor for Ratiometric Imaging of Metal Ions In Vivo Using DNAzymes Conjugated to An Engineered Nano-Luciferase

DNA-based probes have gained significant attention as versatile tools for biochemical analysis, benefiting from their programmability and biocompatibility. However, most existing DNA-based probes rely on fluorescence as the signal output, which can be problematic due to issues like autofluorescence and scattering when applied in complex biological materials such as living cells or tissues. Herein, we report the development of bioluminescent nucleic acid (bioLUNA) sensors that offer laser excitation-independent and ratiometric imaging of the target in vivo. The system is based on computational modelling and mutagenesis investigations of a genetic fusion between circular permutated Nano-luciferase (NLuc) and HaloTag, enabling the conjugation of the protein with a DNAzyme. In the presence of Zn²⁺, the DNAzyme sensor releases the fluorophore-labelled strand, leading to a reduction in bioluminescent resonance energy transfer (BRET) between the luciferase and fluorophore. Consequently, this process induces ratiometric changes in the bioluminescent signal. We demonstrated that this bioLUNA sensor enabled imaging of both exogenous Zn²⁺ in vivo and endogenous Zn²⁺ efflux in normal epithelial prostate and prostate tumors. This work expands the DNAzyme sensors to using bioluminescence and thus has enriched the toolbox of nucleic acid sensors for a broad range of biomedical applications.     

Programmable DNA Nanoflowers for Biosensing,Bioimaging, and Therapeutics

DNA nanostructures have shown excellent prospects in biomedical applications owing to their unique sequence programmability, function designability, and biocompatibility. As a type of unique DNA–inorganic hybrid nanostructures, DNA nanoflowers (DNFs) have attracted considerable attention in the past few years. Precise design of the DNA sequence enables the functions of DNFs to be customized. Specifically, DNFs exhibit high physiological stability and more diverse properties by virtue of the incorporation of inorganic materials, which in turn have been applied in an assortment of biomedical fields. In this review, the design, synthesis, and biomedical applications of programmable DNFs are discussed. First, the background of DNA-based materials and the fundamentals of DNFs are briefly introduced. In the second part, two synthetic methods of DNFs are categorized as the rolling circle amplification and salt aging method, focusing on the formation mechanism of DNFs and differences between the synthetic methods. In the third part, the biomedical applications of DNFs functional materials are summarized, including biosensing, bioimaging, and therapeutics. Finally, the challenges and future opportunities of DNFs are discussed toward more widespread applications.     

Framework Nucleic Acids for Cell Imaging and Therapy

Over the past decade,structural DNA nanotechnology has been well developed to be a promising and powerful technique to generate various nanostructures with programmability,spatial organization and biocompatibi-lity.With the advent of computer-aided tools,framework nucleic acids have been employed in a series of biomedical applications,ranging from biosensing,bioimaging,diagnosis,to therapeutics.In this review,we summarized recent advances in the construction of precisely assembled DNA nanostructures,and DNA-engineered biomimetics.We also outlined the challenges and opportunities for the translational applications of framework nucleic acids.     

CRISPR Mediated Biosensing Toward Understanding Cellular Biology and Point-of-Care Diagnosis

Recent advances in CRISPR based biotechnologies have greatly expanded our capabilities to repurpose CRISPR for the development of biomolecular sensors for diagnosing diseases and understanding cellular pathways. The key attribute that allows CRISPR to be widely utilized is the programmable and highly selective mechanism. In this Minireview, we first illustrate the molecular principle of CRISPR functioning process from sensing to actuating. Next, the CRISPR based biosensing strategies for nucleic acids, proteins and small molecules are summarized. We highlight some of recent advances in applications for in vitro detection of biomolecules and in vivo imaging of cellular networks. Finally, the challenges with, and exciting prospects of, CRISPR based biosensing developments are discussed.     

Synthesis,Characterization and Evaluation of Peptide Nanostructures for Biomedical Applications

There is a challenging need for the development of new alternative nanostructures that can allow the coupling and/or encapsulation of therapeutic/diagnostic molecules while reducing their toxicity and improving their circulation and in-vivo targeting. Among the new materials using natural building blocks, peptides have attracted significant interest because of their simple structure, relative chemical and physical stability, diversity of sequences and forms, their easy functionalization with (bio)molecules and the possibility of synthesizing them in large quantities. A number of them have the ability to self-assemble into nanotubes, -spheres, -vesicles or -rods under mild conditions, which opens up new applications in biology and nanomedicine due to their intrinsic biocompatibility and biodegradability as well as their surface chemical reactivity via amino- and carboxyl groups. In order to obtain nanostructures suitable for biomedical applications, the structure, size, shape and surface chemistry of these nanoplatforms must be optimized. These properties depend directly on the nature and sequence of the amino acids that constitute them. It is therefore essential to control the order in which the amino acids are introduced during the synthesis of short peptide chains and to evaluate their in-vitro and in-vivo physico-chemical properties before testing them for biomedical applications. This review therefore focuses on the synthesis, functionalization and characterization of peptide sequences that can self-assemble to form nanostructures. The synthesis in batch or with new continuous flow and microflow techniques will be described and compared in terms of amino acids sequence, purification processes, functionalization or encapsulation of targeting ligands, imaging probes as well as therapeutic molecules. Their chemical and biological characterization will be presented to evaluate their purity, toxicity, biocompatibility and biodistribution, and some therapeutic properties in vitro and in vivo. Finally, their main applications in the biomedical field will be presented so as to highlight their importance and advantages over classical nanostructures.     

Programmable mismatch-fueled high-efficiency DNA signal amplifier

Herein, by introducing mismatches, a high-efficiency mismatch-fueled catalytic multiple-arm DNA junction assembly (M-CMDJA) with high-reactivity and a high-threshold is developed as a programmable DNA signal amplifier for rapid detection and ultrasensitive intracellular imaging of miRNA. Compared with traditional nucleic acid signal amplification (NASA) with a perfect complement, the M-CMDJA possesses larger kinetic and thermodynamic favorability owing to the more negative reaction standard free energy (ΔG) as driving force, resulting in much higher efficiency and rates. Once traces of the input initiator react with the mismatched substrate DNA, it could be converted into amounts of output multiple-arm DNA junctions via the M-CMDJA as the functional DNA conversion nanodevice. Impressively, the mismatch-fueled catalytic four-arm DNA junction assembly (M-CFDJA) exhibits high conversion efficiency up to 1.05 × 10⁸ in 30 min, which is almost ten times more than those of conventional methods. Therefore, the M-CMDJA could easily address the challenges of traditional methods: slow rates and low efficiency. In application, the M-CFDJA as a DNA signal amplifier was successfully used to develop a biosensing platform for rapid miRNA detection with a LOD of 6.11 aM and the ultrasensitive intracellular imaging of miRNA, providing a basis for the next-generation of versatile DNA signal amplification methods for ultimate applications in DNA nanobiotechnology, biosensing assay, and clinical diagnoses.

We proposed an ingenious mismatch-enhanced catalytic multiple-arm DNA junction assembly (M-CMDJA) which possesses more negative reaction standard free energy (ΔG) as the driving force, resulting in quite high conversion efficiency and much faster reaction speed.     

Recent Development of DNA-modified AIEgen Probes for Biomedical Application

A variety of DNA-based probes are utilized for the detections of multiple analytes and DNA nanotechnology has been thriving for recent decades and achieving numerous nanostructures,mainly focusing on DNA morphology modulation and multifunctional systems engineered into to the complicated works.Among the numerous detections,fluorescence method is a non-invasive,highly selective and sensitive means for varieties of applications,but their emissions are often compromised by the aggregation-caused quenching(ACQ)effect,which weakens their applications.The aggregation induced emission luminogens(AIEgens)are created with non emissive or weakly emissive in a low concentration but emit strong fluorescence in a high concentration with aggregated states.Herein,numerous functionalized AIEgens have been emerged and used for detection and imaging and DNA-modified AIEgen probes are introduced.In this vein,here we report the progress on DNA-modified AIEgen probes in recent years and highlight their conjugation strategies including covalent bonding,electrostatic interaction and their applications of biosensing.Moreover,multiple DNA strands are needed to introduce into the DNA-modified AIEgen probes for more purposes.At the end,some challenges are mentioned to discuss the new trend of DNA-modified AIEgen probes.     

Building multipurpose nano-toolkit by rationally decorating NIR-II fluorophore to meet the needs of tumor diagnosis and treatment

Phototheranostics have attracted tremendous attention in cancer diagnosis and treatment because of the noninvasiveness and promising effectiveness. Developing advanced phototheranostic agents with long emission wavelength, excellent biocompatibility, great tumor-targeting capability, and efficient therapeutic effect is highly desirable. However, the mutual constraint between imaging and therapeutic functions usually hinders their wide applications in biomedical field. To balance this contradiction, we herein rationally designed and synthesized three novel tumor-targeted NIR-II probes (QR-2PEG₃₂₁, QR-2PEG₁₀₀₀, and QR-2PEG₅₀₀₀) by conjugating three different chain lengths of PEG onto an integrin α_vβ₃-targeted NIR-II heptamethine cyanine fluorophore, respectively. In virtue of the essential amphiphilic characteristics of PEG polymers, these probes display various degree of aggregation in aqueous buffer accompanying with differential NIR-II imaging and photothermal (PTT) therapeutic performance. Both in vitro and in vivo results have demonstrated that probe QR-2PEG₅₀₀₀ has the best NIR-II imaging performance with prominent renal clearance, whereas QR-2PEG₃₂₁ possesses excellent photoacoustic signal as well as PTT effect, which undoubtedly provides a promising toolbox for tumor diagnosis and therapy. We thus envision that these synthesized probes have great potential to be explored as a toolkit for precise diagnosis and treatment of malignant tumors.     

Step-growth polymerization of traptavidin-DNA conjugates for plasmonic nanostructures

Here, we use two important biomaterials, protein and DNA, to construct self-assembled linear nanostructures through Watson-Crick base-paring of DNAs. We apply a simple magnetic separation method to purify traptavidin-DNA conjugates, and demonstrate synthesis of linear arrays of traptavidin-DNA conjugates via the step-growth polymerization approach with pre-determined DNA sequences. Using the traptavidin-DNA array as a template, we assemble gold nanoparticles to form linear plasmonic nanostructures in a programmable manner. The traptavidin-DNA conjugates thus provide a convenient platform for one-dimensional assembly of biotinylated nanomaterials for many biomedical applications from drug delivery to bio-sensing.     

DNAzymes for sensing, nanobiotechnology and logic gate applications

Catalytic nucleic acids (DNAzymes or ribozymes) are selected by the systematic evolution of ligands by exponential enrichment process (SELEX). The catalytic functions of DNAzymes or ribozymes allow their use as amplifying labels for the development of optical or electronic sensors. The use of catalytic nucleic acids for amplified biosensing was accomplished by designing aptamer-DNAzyme conjugates that combine recognition units and amplifying readout units as in integrated biosensing materials. Alternatively, "DNA machines" that activate enzyme cascades and yield DNAzymes were tailored, and the systems led to the ultrasensitive detection of DNA. DNAzymes are also used as active components for constructing nanostructures such as aggregated nanoparticles and for the activation of logic gate operations that perform computing.     

Recent progress on DNA block copolymer

Organic polymers are combined with DNA resulting DNA block copolymers (DBCs) that can simultaneously show the properties of the polymer and DNA. We will discuss some examples of recent developments in the syntheses, structure manipulations, and applications of DBCs.     

Novel photo-theranostic GdB6 nanoparticles for fluorescence imaging and NIR-photothermal therapy

The development of multifunctional theranostic nano-agents is an important resolution for personalized treatment of cancer. In this work, we synthesized a new kind of gadolinium boride nanoparticles (GBN) by a microwave-assisted chemical etching method, and discovered their optical characteristics including fluorescence imaging and near-infrared (NIR) photothermal conversion capability. Bright greenishyellow fluorescence enabled for intracellular localization, while effective NIR-photothermal conversion supported photothermal therapy (PTT). In vitro and in vivo results indicated that GBN exhibited a superior antitumor performance and high biocompatibility. This study demonstrated a promising multifunctional theranostic nanoplatform for cancer treatment.     

Enzymatic fabrication of DNA nanostructures: extension of a self-assembled oligonucleotide monolayer on gold arrays

Nucleic acid nanostructures are useful as templates for bionanofabrication of composite molecular nanostructures in materials science, molecular electronics, and biosensing. Here, we demonstrate that terminal deoxynucleotidyl transferase, which repetitively adds mononucleotides to the 3' end of a short DNA initiator, can be used to rapidly fabricate DNA nanostructures up to 121 nm high with lateral dimensions from 0.1 to 4 mum in 2 h. These programmable scaffolds can potentially be employed to build more complex nanostructures consisting of natural or unnatural nucleotides with selective docking sites along the single-stranded DNA.