Nucleic Acid Framework-Enabled Spatial Organization for Biological Applications

He Z., Xiang W., Fan Q., Wang L., Chao J.

A rectangle DNA origami nanostructure equipped with doxorubicin -derived prodrugs targeting tumor cell-specific enzyme (NQO1) is constructed. Combining high prodrug payload of DNA origami and NQO1-activated chemotherapy, this nanosystem presents...

Knappe G.A., Wamhoff E., Bathe M.

DNA origami has emerged as a powerful method to generate DNA nanostructures with dynamic properties and nanoscale control. These nanostructures enable complex biophysical studies and the fabrication of next-generation therapeutic devices. For these applications, DNA origami typically needs to be functionalized with bioactive ligands and biomacromolecular cargos. Here, we review methods developed to functionalize, purify and characterize DNA origami nanostructures. We identify remaining challenges such as limitations in functionalization efficiency and characterization. We then discuss where researchers can contribute to further advance the fabrication of functionalized DNA origami. DNA origami nanostructures are useful constructs for biophysical and therapeutic studies. This Review discusses how these nanostructures are functionalized with bioactive conjugates, purified and characterized, and compares the advantages and limitations of these methods in the context of different applications.

Wang Y., Feng H., Huang K., Quan J., Yu F., Liu X., Jiang H., Wang X.

A signal amplification sensing system with target-triggered DNA cascade reaction combined with dual-signal readout technology was designed for ultrasensitive analysis of miRNA. The highly conductive metal organic frameworks (MOFs) derivative, N-doped carbon dodecahedron (N-PCD) was deposited with gold nanoparticles as the electrode substrate, which could assist the electron transfer between the molecular probe and the electrode surface, and could remarkably enhance electrochemical response. Tetrahedral DNA nanostructure (T4-DNA) with high structural stability and mechanical stiffness was designed to improve the loading capacity and binding efficiency of the target, thus increasing the sensitivity of the system. The non-enzymatic amplification method based on the DNA cascade reaction allows the electrochemical responses from dual signal DNA probes labeled with ferrocene (Fc) and methylene blue (MB), respectively in turn to improve the reliability of detection. Under optimal conditions, the sensor has a linear range of 5-1.0 × 104 fM, and the limit of detection is as low as 1.92 fM and 3.74 fM for Fc and MB labeled probe, respectively. This strategy raises the promising application for the rapid detection of miRNA targets with low abundance in complex biological systems.

Zhang J., Chen M., Peng Y., Li S., Han D., Ren S., Qin K., Li S., Han T., Wang Y., Gao Z.

• Molecular dynamics simulation of the CTNAT and the ATNAT reporter. • Multi-channel detection and background signal of the ATNAT and the CTNAT reporter. • Redesigns of the CTNAT reporter in the nanosize and the hybridization efficiency. • EXPAR-cDNA-CTNAT strategy for the detection of three fatigue biomarkers. • Accurate, sensitive and specific fatigue diagnosis. The nucleic tweezers-based detection strategies have shown excellent application prospects in molecular detection and diagnosis thanks to the specific target responsiveness and good biocompatibility. However, the limited detection efficiency and unsatisfactory sensitivity make their applications in complex diagnosis difficult, and few reports focused on it. Here, we explored the molecular dynamics simulations and biosensing performance of the Tetrahedral Nucleic Acid Tweezers, including the Antennae like-Tetrahedron Nucleic Acid Tweezer (ATNAT) and the Covered-Tetrahedron Nucleic Acid Tweezer (CTNAT), revealing the molecular dynamics of ATNAT and CTNAT reporters visually and molecularly. After that, we compared the multi-target detection capabilities of DNA tetrahedral tweezers, and combined the CTNAT reporter with better multi-target detection performance with the aptamer and Exponential amplification reaction (EXPAR), developing an efficient and sensitive EXPAR-cDNA-CTNAT strategy. Then we applied the EXPAR-cDNA-CTNAT strategy to detect testosterone, cortisol, and creatine kinase isoenzymes, realizing sensitive and accurate fatigue diagnosis. Compared with the traditional detection strategies, the EXPAR-cDNA-CTNAT strategy showed improved sensitivity and detection efficiency with excellent specificity, and the limits of detection (LODs) for the multi-target detection were as low as 41, 68, and 8 pM, respectively. The EXPAR-cDNA-CTNAT strategy was reliable for multi-target detection, which had great potential in biological science, food safety, and medical diagnosis.

Safieddine A., Coleno E., Lionneton F., Traboulsi A., Salloum S., Lecellier C., Gostan T., Georget V., Hassen-Khodja C., Imbert A., Mueller F., Walter T., Peter M., Bertrand E.

The ability to visualize RNA in its native subcellular environment by using single-molecule fluorescence in situ hybridization (smFISH) has reshaped our understanding of gene expression and cellular functions. A major hindrance of smFISH is the difficulty to perform systematic experiments in medium- or high-throughput formats, principally because of the high cost of generating the individual fluorescent probe sets. Here, we present high-throughput smFISH (HT-smFISH), a simple and cost-efficient method for imaging hundreds to thousands of single endogenous RNA molecules in 96-well plates. HT-smFISH uses RNA probes transcribed in vitro from a large pool of unlabeled oligonucleotides. This allows the generation of individual probes for many RNA species, replacing commercial DNA probe sets. HT-smFISH thus reduces costs per targeted RNA compared with many smFISH methods and is easily scalable and flexible in design. We provide a protocol that combines oligo pool design, probe set generation, optimized hybridization conditions and guidelines for image acquisition and analysis. The pipeline requires knowledge of standard molecular biology tools, cell culture and fluorescence microscopy. It is achievable in ~20 d. In brief, HT-smFISH is tailored for medium- to high-throughput screens that image RNAs at single-molecule sensitivity. In HT-smFISH, multiple RNA probes are generated by parallel in vitro transcription from a large pool of unlabeled oligonucleotides. This reduces costs per targeted RNA compared with many smFISH methods and is easily scalable and flexible in design.

Bialy R.M., Mainguy A., Li Y., Brennan J.D.

Functional nucleic acids (FNAs), including DNA aptamers and DNAzymes, are finding increasing use as molecular recognition elements for point-of-care (POC) assays and sensors. An ongoing challenge in the development of FNA-based POC sensors is the ability to achieve detection of low levels of analyte without compromising assay time and ease of use. Rolling circle amplification (RCA) is a leading nucleic acid (NA) isothermal amplification method which can be coupled with FNAs for the ultrasensitive detection of non-NA targets. Herein we examine the key considerations required when designing FNA-coupled biosensors utilizing RCA. Specifically, we describe methods for using FNAs as inputs to regulate RCA, various modes of RCA amplification, and methods to detect the output of the RCA reaction, along with how these can be combined to allow detection of non-NA targets. Recent progress on development of portable optical and electrochemical POC devices that incorporate RCA is then described, followed by a summary of key challenges and opportunities in the field.

Wang Y., Zhu J., Jia W., Xiong H., Qiu W., Xu R., Lin Y.

Alzheimer's disease is a neurodegenerative disease caused by excessive amyloid β protein-induced neurotoxicity. However, drugs targeting amyloid β protein production face many problems, such as the low utilization rate of drugs by cells and the difficulty of drugs in penetrating the blood-brain barrier. A tetrahedral framework nucleic acid is a new type of nanonucleic acid structure that functions as a therapy and drug carrier. Here, we synthesized a BACE1 aptamer-modified tetrahedral framework nucleic acid and tested its therapeutic effect on Alzheimer's disease in vitro and in vivo. Our results demonstrated that the tetrahedral framework nucleic acid could be used as a carrier to deliver the BACE1 aptamer to the brain to reduce the production of amyloid β proteins. It also played an antiapoptotic role by reducing the production of reactive oxygen species. Thus, this nanomaterial is a potential drug for Alzheimer's disease.

Wan S., Liu S., Sun M., Zhang J., Wei X., Song T., Li Y., Liu X., Chen H., Yang C.J., Song Y.

Natural ligand-receptor interactions that play pivotal roles in biological events are ideal models for design and assembly of artificial recognition molecules. Herein, aiming at the structural characteristics of the spike trimer and infection mechanism of SARS-CoV-2, we have designed a DNA framework-guided spatial-patterned neutralizing aptamer trimer for SARS-CoV-2 neutralization. The ∼5.8 nm tetrahedral DNA framework affords precise spatial organization and matched valence as four neutralizing aptamers (MATCH-4), which matches with nanometer precision the topmost surface of SARS-CoV-2 spike trimer, enhancing the interaction between MATCH-4 and spike trimer. Moreover, the DNA framework provides a dimensionally complementary nanoscale barrier to prevent the spike trimer-ACE2 interaction and the conformational transition, thereby inhibiting SARS-CoV-2-host cell fusion and infection. As a result, the spatial- and valence-matched MATCH-4 ensures improved binding affinity and neutralizing activity against SARS-CoV-2 and its varied mutant strains, particularly the current Omicron variant, that are evasive of the majority of existing neutralizing antibodies. In addition, because neutralizing aptamers specific to other targets can be evolved and assembled, the present design has the potential to inhibit other wide-range and emerging pathogens.

Rajwar A., Shetty S.R., Vaswani P., Morya V., Barai A., Sen S., Sonawane M., Bhatia D.

Fabrication of nanoscale DNA devices to generate 3D nano-objects with precise control of shape, size, and presentation of ligands has shown tremendous potential for therapeutic applications. The interactions between the cell membrane and different topologies of 3D DNA nanostructures are crucial for designing efficient tools for interfacing DNA devices with biological systems. The practical applications of these DNA nanocages are still limited in cellular and biological systems owing to the limited understanding of their interaction with the cell membrane and endocytic pathway. The correlation between the geometry of DNA nanostructures and their internalization efficiency remains elusive. We investigated the influence of the shape and size of 3D DNA nanostructures on their cellular internalization efficiency. We found that one particular geometry, i.e., the tetrahedral shape, is more favored over other designed geometries for their cellular uptake in 2D and 3D cell models. This is also replicable for cellular processes like cell invasion assays in a 3D spheroid model, and passing the epithelial barriers in in vivo zebrafish model systems. Our work provides detailed information for the rational design of DNA nanodevices for their upcoming biological and biomedical applications.

Li J., Lai Y., Li M., Chen X., Zhou M., Wang W., Li J., Cui W., Zhang G., Wang K., Liu L., Lin Y.

• Tetrahedral DNA nanostructure provides an effective drug delivery vehicle. • We constructed a 3D-bioprinted scaffold loaded with TDN-clindamycin complex. • The novel scaffold was biocompatible, with osteogenic and antimicrobial properties. • The scaffold significantly improved repair in rat model of infected bone defect. Infection of bone defects is a common clinical problem that negatively impacts bone repair and may result in the development of antibiotic resistance due to the long-term use of antibiotics. To the best of our knowledge, integrating tetrahedral DNA nanostructure (TDN) drug delivery with 3D bioprinting has not been reported to treat infected bone defects. However, numerous studies have focused on effectively controlling infected bone defects and achieving functional reconstruction. In this study, TDN was proposed as a drug delivery vehicle to enhance cell penetration and the antibacterial properties of clindamycin (CLI), a common antibiotic used to treat osteomyelitis. A 3D hybrid scaffold loaded with TDN-CLI complexes was constructed using bioprinting technology. Its structure and biological properties were characterized, and the scaffold was further applied to treat methicillin-resistant Staphylococcus aureus (MRSA) infection in a rat model of bone defect. The results demonstrated that the TDN-CLI-loaded 3D bioprinted hybrid scaffold possessed excellent biocompatibility, outstanding osteogenic and antimicrobial activity, and significantly improved the repair of infected bone defects in the rat model. The TDN-CLI-loaded hybrid scaffold developed in the current study has broad application prospects for treating infected bone defects.

Zhang W., Liu N., Zhang J.

Coronavirus Disease 2019 (COVID-19), which poses an extremely serious global impact on human public healthcare, represents a high transmission and disease-causing viral infection caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that is expanding at a rapid pace. Therefore, it is urgent for researchers to establish effective platforms for the assay and treatment of SARS-CoV-2. Functional nucleic acids (FNAs), comprising aptamers and nucleases, are of primary concern within the biological and medical communities owing of the distinctive properties of their target recognition and catalysis. This review will concentrate on the essential aspects of insights regarding FNAs and their technological expertise for the diagnostic and therapeutic utilization against COVID-19. We first offer a historical perspective of the COVID-19 pandemics, its clinical characteristics and potential biomarkers. Then, we briefly discuss the current diagnostic and therapeutic methodology towards COVID-19, highlighting the superiorities and existing shortcomings. After that, we introduce the key features of FNAs, and summarize recent progress of in vitro selection of FNAs for SARS-CoV-2 specific proteins and RNAs, followed by highlighting the general concept of translating FNAs into functional probes for diagnostic and therapeutic purposes. Then, we critically review the emerging FNAs-based diagnostic and therapeutic strategies that are fast, precise, efficient, and highly specific to fight COVID-19. Finally, we identify remaining challenges and offer future outlook of this emerging field.

Liu Y., Teng L., Yin B., Meng H., Yin X., Huan S., Song G., Zhang X.

Photoacoustic (PA) imaging technology, a three-dimensional hybrid imaging modality that integrates the advantage of optical and acoustic imaging, has great application prospects in molecular imaging due to its high imaging depth and resolution. To endow PA imaging with the ability for real-time molecular visualization and precise biomedical diagnosis, numerous activatable molecular PA probes which can specifically alter their PA intensities upon reacting with the targets or biological events of interest have been developed. This review highlights the recent developments of activatable PA probes for precise biomedical applications including molecular detection of the biotargets and imaging of the biological events. First, the generation mechanism of PA signals will be given, followed by a brief introduction to contrast agents used for PA probe design. Then we will particularly summarize the general design principles for the alteration of PA signals and activatable strategies for developing precise PA probes. Furthermore, we will give a detailed discussion of activatable PA probes in molecular detection and biomedical imaging applications in living systems. At last, the current challenges and outlooks of future PA probes will be discussed. We hope that this review will stimulate new ideas to explore the potentials of activatable PA probes for precise biomedical applications in the future.

Wang J., Zhu L., Li T., Li X., Huang K., Xu W.

Functional nucleic acids (FNAs) have emerged as superior molecular recognition elements with high binding affinity, specificity, and catalytic activity. Due to multiple functionalities like specific recognition, stimulus responsiveness, sequence programmability, structural tailorability, and ease of modification, FNAs have been integrated into lateral flow assays (LFAs) to construct simple and sustainable paper-based chromatographic biosensors for rapid, antibody-free, cost-effective, and user-friendly target detection. This review presented a systematic, comprehensive summary of functional nucleic acid-based lateral flow assays (FNA-LFAs). First, the screening, characterization, and tailoring of FNAs to meet the requirements of LFAs were briefly introduced. Second, the multi-functionality of FNAs for developing high-performance LFAs is presented, along with the entire LFA process from sample pretreatment to signal transduction. Finally, the applications of FNA-LFAs in point-of-care testing (POCT) are illustrated along with future research perspectives, aiming to provide a valuable reference for researchers in the field of FNA sensing. • Provided a systematic and comprehensive summary of FNA-LFAs. • Listed and further analyzed six types of FNAs applied in LFAs. • Highlighted the multiple functionalities of FNAs for developing high-performance LFAs. • Dissected the entire LFA construction process step-by-step. • Introduced the primary FNA-LFA applications areas in POCT in detail.

Zhao F., Xie S., Li B., Zhang X.

As significant components of the organism, carbohydrates and glycoconjugates play indispensable roles in energy supply, cell signaling, immune modulation, and tumor cell invasion, and function as biomarkers since aberrance of them has been proved to be associated with the emergence and development of certain diseases. Functional nucleic acids (FNAs) have properties including easy-to-synthesize, good stability, good biocompatibility, low cost, and high programmability, they have attracted significant research attention and been incorporated into biosensors for detecting disease-related carbohydrates and glycoconjugates. This review summarizes the construction strategies and biosensing applications of FNAs-based biosensors in glycobiology in terms of target recognition and signal transduction. By illustrating the mechanisms and comparing the performances, the challenges and development opportunities in this area have been critically elaborated. We believe that this review will provide a better understanding of the role of FNAs in the analysis of disease-related carbohydrates and glycoconjugates, and inspire further discovery in fields that include glycobiology, chemical biology, clinical diagnosis, and drug development.

Rodrigues L.R., Sousa D.A., Carneiro M., Ferreira D., Moreira F.T., Sales M.G.

Abstract: An early diagnosis has the potential to greatly decrease cancer mortality. For that purpose, specific cancer biomarkers have been molecularly targeted by aptamer sequences to enable an accurate and rapid detection. Aptamer-based biosensors for cancer diagnostics are a promising alternative to those using antibodies, due to their high affinity and specificity to the target molecules and advantageous production. Synthetic nucleic acid aptamers are generated by in vitro Systematic Evolution of Ligands by Exponential enrichment (SELEX) methodologies that have been improved over the years to enhance the efficacy and shorten the selection process. Aptamers have been successfully applied in electrochemical, optical, photoelectrochemical and piezoelectrical-based detection strategies. These aptasensors comprise a sensitive, accurate and inexpensive option for cancer detection being used as point-of-care devices. This review highlights the recent advances in cancer biomarkers, achievements and optimizations made in aptamer selection, as well as the different aptasensors developed for the detection of several cancer biomarkers.

Niu Y., Chen B., Zhang H., Zheng W., Wu J., Yang L., Yang M., Yu H.