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Understanding the structural reconfiguration of a biomolecular receptor remains a topic of particular interest to the biosensing community. This is because conformationally changing receptors are commonly employed in biosensors to harness their capability to bind specifically to their target. Often, such receptors are attached to surfaces so that binding can be transduced into a measurable response. Doing so, however, can impose constraints on the possible configurations they can adopt. Such constraints can ultimately influence, for example, their receptor-target binding models or their affinity, which is essential to provide the desired analytical performances in biosensors. Motivated by the idea of gaining further insights into the impact of surface attachment on conformationally switching receptors attached to surfaces, we explore here the various surface-based techniques capable of monitoring structural changes. We decided to narrow our survey to techniques that have been applied to the investigation of nucleic acids to provide an overview of their key features. We envision that this will bring a broader perspective of the field and the challenges ahead with the hopes of "finding the switch" in surface-attached biomolecular receptors.

The ability to dive deep into specific rare cell populations is critical for understanding tissue physiology and pathology across various biological domains. As single-cell RNA-seq flourishes, many newly discovered cell subtypes are defined by their transcriptomic markers. However, our ability to retrieve and analyze cells based on their nucleic acid markers remains underdeveloped. Here, we present Double Emulsion PCR-Initiated Cell sorting and Transcriptomic Sequencing (DEPICT-seq), a high-throughput droplet nucleic acid cytometry method that integrates batch cell fixation for cellular information preservation, double emulsion digital PCR-based cell sorting to target nucleic acid markers of interest, and in-depth full-length transcriptomic analyses at single-cell resolution. We utilize DEPICT-seq to isolate and characterize T cell receptor (TCR)-engineered T cells within a mixed population and also demonstrate a variation of the workflow by incorporating an RNase H-dependent PCR step to enrich full-length TCR sequences for paired single-cell TCR sequencing and transcriptomic profiling.

Lipid nanoparticles (LNPs) are gaining recognition as potentially effective carriers for delivery of therapeutic agents, including nucleic acids (DNA and RNA), for the prevention and treatment of various diseases. Much effort has been devoted to the implementation of microfluidic techniques for the production of monodisperse and stable LNPs and the improvement of encapsulation efficiency. Here, we developed three-dimensional (3D)-printed ring micromixers for the production of size-controllable and monodispersed LNPs with a high mRNA delivery efficiency. The effects of flow rate and ring shape asymmetry on the mixing performance were initially examined. Furthermore, the physicochemical properties (such as hydrodynamic diameter, polydispersity, and encapsulation efficiency) of the generated LNPs were quantified as a function of these physical parameters via biochemical analysis and cryo-electron microscopy imaging. With a high production rate of 68 mL/min, our 3D-printed ring micromixers can be used to manufacture LNPs with diameters less than 90 nm, low polydispersity (<0.2), and high mRNA encapsulation efficiency (>91%). Despite the simplicity of the ring-shaped mixer structure, we can produce mRNA-loaded LNPs with exceptional quality and high throughput, outperforming costly commercial micromixers.

Periodontitis, with its persistent nature, causes significant distress for most sufferers. Current treatments, such as mechanical cleaning and surgery, often fail to fully address the underlying overactivation of fibroblasts that drives this degradation. Targeting the post-transcriptional regulation of fibroblasts, particularly at the 3'-untranslated regions (3'UTR) of pathogenic genes, offers a therapeutic strategy for periodontitis. Herein, we developed a DNA nanorobot for this purpose. This system uses a dynamic DNA nanoframework to incorporate therapeutic microRNAs through molecular recognition and covalent bonds, facilitated by DNA monomers modified with disulfide bonds. The assembled-DNA nanoframework is encapsulated in a cell membrane embedded with a fibroblast-targeting peptide. By analyzing the 3'UTR regions of pathogenic fibroblast genes FOSB and JUND , we identified the therapeutic microRNA as miR-1-3p and integrated it into this system. As expected, the DNA nanorobot delivered the internal components to fibroblasts by the targeting peptide and outer membrane that responsively releases miR-1-3p under intracellular glutathione. It resulted in a precise reduction of mRNA and suppression of protein function in pathogenic genes, effectively reprogramming fibroblast behavior. Our results confirm that this approach not only mitigates the inflammation but also promotes tissue regeneration in periodontal models, offering a promising therapeutic avenue for periodontitis.

Biopharmaceuticals such as nucleic acids, proteins, and peptides constitute a new array of treatment modalities for chronic ailments. Invasive routes remain the mainstay of administering biopharmaceuticals due to their labile nature in the biological environment. However, it is not preferred for long-term therapy due to the lack of patient adherence and clinical suitability. Therefore, alternative routes of administration are sought to utilize novel biopharmaceutical therapies to their utmost potential. Nanoparticle-mediated pulmonary delivery of biologics can facilitate both local and systemic disorders. Solid lipid nanoparticles (SLNs) afford many opportunities as pulmonary carriers due to their physicochemical stability and ability to incorporate both hydrophilic and hydrophobic moieties, thus allowing novel combinatorial drug/gene therapies. These applications include pulmonary infections, lung cancer, and cystic fibrosis, while systemic delivery of biomolecules, like insulin, is also attractive for the treatment of chronic ailments. This Review explores physiological and particle-associated factors affecting pulmonary delivery of biopharmaceuticals. It compares the advantages and limitations of SLNs as pulmonary nanocarriers along with design improvements underway to overcome these limitations. Current research illustrating various SLN designs to deliver proteins, peptides, plasmids, oligonucleotides, siRNA, and mRNA is also summarized.

Nucleic acid-based electrochemical sensors (NBEs) use oligonucleotides as affinity reagents for the detection of a variety of targets, ranging from small-molecule therapeutics to whole viruses. Because of their versatility in molecular sensing, NBEs are being developed broadly for diagnostic and biomedical research applications. Benchmark NBEs are fabricated via self-assembly of thiol-based monolayers on gold. Although robust for rapid prototyping, thiol monolayers suffer from limitations in terms of stability under voltage modulation and in the face of competitive ligands such as thiolated molecules naturally occurring in biofluids. Additionally, gold cannot be deployed as an NBE substrate for all biomedical applications, such as in cases where molecular measurements coupled to real-time, under-the-sensor tissue imaging is needed. Seeking to overcome these limitations, the field of NBEs is pursuing alternative ligands and electrode surfaces. In this perspective, I discuss new interface fabrication strategies that have successfully achieved NBE sensing, or that have the potential to allow NBE sensing on conductive surfaces other than gold. I hope this perspective will provide the reader with a fresh view of how future NBE interfaces could be constructed and will serve as inspiration for the pursuit of collaborative developments in the field of NBEs.

The role of nucleic acids in protein folding and aggregation is an area of continued research, with relevance to understanding both basic biological processes and disease. In this review, we provide an overview of the trajectory of research on both nucleic acids as chaperones and their roles in several protein misfolding diseases. We highlight key questions that remain on the biophysical and biochemical specifics of how nucleic acids have large effects on multiple proteins' folding and aggregation behavior and how this pertains to multiple protein misfolding diseases.

Plant synthetic biology is applied in sustainable agriculture, clean energy, and biopharmaceuticals, addressing crop improvement, pest resistance, and plant-based vaccine production by introducing exogenous genes into plants. This technique faces challenges delivering genes due to plant cell walls and intact cell membranes. Novel approaches are required to address this challenge, such as utilizing nanomaterials known for their efficiency and biocompatibility in gene delivery. This work investigates metal-organic frameworks (MOFs) for gene delivery in intact plant cells by infiltration. Hence, small-sized ZIF-8 nanoparticles (below 20 nm) were synthesized and demonstrated effective DNA/RNA delivery into Nicotiana benthamiana leaves and Arabidopsis thaliana roots, presenting a promising and simplified method for gene delivery in intact plant cells. We further demonstrate that small-sized ZIF-8 nanoparticles protect RNA from RNase degradation and successfully silence an endogenous gene by delivering siRNA in N. benthamiana leaves.

Chirality, one of the most fundamental properties of natural molecules, plays a significant role in biochemical reactions. Nanomaterials with chiral characteristics have superior properties, such as catalytic properties, optoelectronic properties, and photothermal properties, which have significant potential for specific applications in nanomedicine. Biomolecular modifications such as nucleic acids, peptides, proteins, and polysaccharides are sources of chirality for nanomaterials with great potential for application in addition to intrinsic chirality, artificial macromolecules, and metals. Two-dimensional (2D) nanomaterials, as opposed to other dimensions, due to proper surface area, extensive modification sites, drug loading potential, and simplicity of preparation, are prepared and utilized in diagnostic applications, drug delivery research, and tumor therapy. Current advanced studies on 2D chiral nanomaterials for biomedicine are focused on novel chiral development, structural control, and materials sustainability applications. However, despite the advances in biomedical research, chiral 2D nanomaterials still confront challenges such as the difficulty of synthesis, quality control, batch preparation, chiral stability, and chiral recognition and selectivity. This review aims to provide a comprehensive overview of the origins, synthesis, applications, and challenges of 2D chiral nanomaterials with biomolecules as cargo and chiral modifications and highlight their potential roles in biomedicine.

Listeria monocytogenes (LM) is one of the most invasive foodborne pathogens that cause listeriosis, making it imperative to explore novel inhibiting strategies for alleviating its infection. The adhesion and invasion of LM within host cells are partly orchestrated by an invasin protein internalin A (InlA), which facilitates bacterial passage by interacting with the host cell E-cadherin (E-Cad). Hence, in this work, we proposed an aptamer blocking strategy by binding to the region on InlA that directly mediated E-Cad receptor engagement, thereby alleviating LM infection. An aptamer GA8 with a robust G-quadruplex (G4) structural feature was designed through truncation and base mutation from the original aptamer A8. The molecular docking and dynamics analysis showed that the InlA/aptamer GA8 binding interface was highly overlapping with the natural InlA/E-Cad binding interface, which confirmed that GA8 can tightly and stably bind InlA and block more distinct epitopes on InlA that involved the interaction with E-Cad. On the cellular level, it was confirmed that GA8 effectively blocked LM adhesion with an inhibition rate of 78%. Overall, the robust G4 aptamer-mediated design provides a new direction for the development of inhibitors against other wide-ranging and emerging pathogens.

Public healthcare demands effective and pragmatic diagnostic tools to address the escalating challenges in infection management in resource-limited areas. Recent advances in clustered regularly interspaced short palindromic repeat (CRISPR)-based biosensing promise the development of next-generation tools for disease diagnostics, including point-of-care (POC) testing for infectious diseases. The currently prevailing strategy of developing CRISPR/Cas-based diagnostics exploits only the target identification and trans -cleavage activity of a CRISPR-Cas12a/Cas13a system to provide diagnostic results, and they need to be combined with an additional preamplification reaction to enhance sensitivity. In contrast to this dual-function strategy, here, we present a new approach that collaboratively integrates the triple functions of CRISPR-Cas12a: target identification, sequence-specific enrichment, and signal generation. With this approach, we develop a nucleic acid assay termed Solid-Phase Extraction and Enhanced Detection Assay integrated by CRISPR-Cas12a (SPEEDi-CRISPR) that negates the need for preamplification but significantly improves the detection of limit (LOD) from the pM to fM level. Specifically, using Cas12a-coated magnetic beads, this assay combines efficient solid-phase extraction and enrichment of DNA targets enabled by the sequence-specific affinity of CRISPR-Cas12a with fluorogenic detection by activated Cas12a on beads. SPEEDi-CRISPR, for the first time, leverages the possibility of employing CRISPR/Cas12a in nucleic acid extraction and integrates the ability of both enrichment and detection of CRISPR/Cas into a single platform. Our proof-of-concept studies revealed that the SPEEDi-CRISPR assay has great specificity to distinguish HPV-18 from HPV-16, and Parvovirus B19, in addition to being able to detect HPV-18 at a concentration as low as 2.3 fM in 100 min and 4.7 fM in 60 min. Furthermore, we proved that this assay can be coupled with two point-of-care testing strategies: the smartphone-based fluorescence detector and the lateral flow assay. Overall, these results suggested that our assay could pave a new way for developing CRISPR diagnostics.

Interferon-γ (IFN-γ) release assays (IGRAs) are constrained by the limited diagnostic performance of a single indicator and the excessive Mycobacterium tuberculosis ( Mtb ) antigen stimulation time. This study presents a simultaneous, homogeneous, rapid, and ultrasensitive fluorescence quantification strategy for IFN-γ and IFN-γ-induced protein 10 (IP-10). This method relies on the high-affinity binding of aptamers to IFN-γ and IP-10, the enzyme-free catalytic hairpin assembly reaction, and the heightened sensitivity of CdTe quantum dots to Ag + and hairpin structure C-Ag + -C and carbon dots to Hg 2+ and hairpin structure T-Hg 2+ -T. Under optimized conditions, the selectivity of IFN-γ and IP-10 was excellent, with a linear range spanning from 1 to 100 ag/mL and low limits of detection of 0.3 and 0.5 ag/mL, respectively. Clinical practicality was confirmed through testing of 57 clinical samples. The dual-indicator combination detection showed 92.8% specificity and 93.1% sensitivity, with an area under the curve of 0.899, representing an improvement over the single-indicator approach. The Mtb antigen stimulation time was reduced to 8 h for 6/7 clinical samples. These findings underscore the potential of our approach to enhance the efficiency and performance of a tuberculosis (TB) clinical diagnosis.

Circulating tumor DNA (ctDNA), as a next-generation tumor marker, enables early screening and monitoring of cancer through noninvasive testing. Exploring the development of new methods for ctDNA detection is an intriguing study. In this work, a unique electrochemical biosensor for the ctDNA detector was constructed in the first utilizing Fe single-atom nanozymes-carbon dots (SA Fe-CDs) as a signaling carrier in collaboration with a DNA walker cascade amplification strategy triggered by nucleic acid exonuclease III (Exo III). The electrochemical active surface area of AuNPs/rGO modified onto a glassy carbon electrode (AuNPs/rGO/GCE) was about 1.43 times that of a bare electrode (bare GCE), with good electrical conductivity alongside a high heterogeneous electron transfer rate (5.81 × 10 -3 cm s -1 ), that is, as well as the ability to load more molecules. Sequentially, the DNA walker cascade amplification strategy driven by Exo III effectively converted the target ctDNA into an amplified biosignal, ensuring the sensitivity and specificity of ctDNA. Ultimately, the electrochemical signal was further amplified by introducing SA Fe-CDs nanozymes, which could serve as catalysts for 3,3',5,5'-tetramethylbenzidine (TMB) oxidation with facile responding ( V max = 0.854 × 10 -6 M s -1 ) and robust annexation ( K m = 0.0069 mM). The integration of the triple signal amplification approach achieved detection limits as low as 1.26 aM ( S / N = 3) for a linearity spanning from 5 aM to 50 nM. In this regard, our proposal for a biosensor with exceptional assay properties in complicated serum environments had great potential for early and timely diagnosis of cancer.

Predicting the protein-nucleic acid (PNA) binding affinity solely from their sequences is of paramount importance for the experimental design and analysis of PNA interactions (PNAIs). A large number of currently developed models for binding affinity prediction are limited to specific PNAIs while also relying on the sequence and structural information of the PNA complexes for both training and testing, and also as inputs. As the PNA complex structures available are scarce, this significantly limits the diversity and generalizability due to the small training data set. Additionally, a majority of the tools predict a single parameter, such as binding affinity or free energy changes upon mutations, rendering a model less versatile for usage. Hence, we propose DeePNAP, a machine learning-based model built from a vast and heterogeneous data set with 14,401 entries (from both eukaryotes and prokaryotes) from the ProNAB database, consisting of wild-type and mutant PNA complex binding parameters. Our model precisely predicts the binding affinity and free energy changes due to the mutation(s) of PNAIs exclusively from their sequences. While other similar tools extract features from both sequence and structure information, DeePNAP employs sequence-based features to yield high correlation coefficients between the predicted and experimental values with low root mean squared errors for PNA complexes in predicting K D and ΔΔ G , implying the generalizability of DeePNAP. Additionally, we have also developed a web interface hosting DeePNAP that can serve as a powerful tool to rapidly predict binding affinities for a myriad of PNAIs with high precision toward developing a deeper understanding of their implications in various biological systems. Web interface: http://14.139.174.41:8080/.

The development of receptors suitable for the continuous detection of analytes in complex, interferent-rich samples remains challenging. Antibodies are highly sensitive but difficult to engineer in order to introduce signaling functionality, while aptamer switches are easy to construct but often yield only a modest target sensitivity. We present here a programmable antibody and DNA aptamer switch (PANDAS), which combines the desirable properties of both receptors by using a nucleic acid tether to link an analyte-specific antibody to an internal strand-displacement (ISD)-based aptamer switch that recognizes the same target through different epitopes. The antibody increases PANDAS analyte binding due to its high affinity, and the effective concentration between the two receptors further enhances two-epitope binding and fluorescent aptamer signaling. We developed a PANDAS sensor for the clotting protein thrombin and show that a tuned design achieves a greater than 300-fold enhanced sensitivity compared to that of using an aptamer alone. This design also exhibits reversible binding, enabling repeated measurements with a temporal resolution of ∼10 min, and retains excellent sensitivity even in interferent-rich samples. With future development, this PANDAS approach could enable the adaptation of existing protein-binding aptamers with modest affinity to sensors that deliver excellent sensitivity and minute-scale resolution in minimally prepared biological specimens.

Solid-state (SS-) nanopore sensing has gained tremendous attention in recent years, but it has been constrained by its intrinsic lack of selectivity. To address this, we previously established a novel SS-nanopore assay that produces translocation signals only when a target biotinylated nucleic acid fragment binds to monovalent streptavidin (MS), a protein variant with a single high-affinity biotin-binding domain. While this approach has enabled selective quantification of diverse nucleic acid biomarkers, sensitivity enhancements are needed to improve the detection of low-abundance translational targets. Because the translocation dynamics that determine assay efficacy are largely governed by constituent charge characteristics, we here incorporate a polyhistidine-tagged MS (hMS) to alter the component detectability. We investigate the effects of buffer pH, salt concentration, and SS-nanopore diameter on the performance with the alternate reagent, achieve significant improvements in measurement sensitivity and selectivity, and expand the range of device dimensions viable for the assay. We used this improvement to detect as little as 1 nM miRNA spiked into human plasma. Overall, our findings improve the potential for broader applications of SS-nanopores in the quantitative analyses of molecular biomarkers.

G-quadruplexes (G4s) are nucleic acid structures crucial for the regulation of gene expression and genome maintenance. While they hold promise as nanodevice components, achieving desired G4 folds requires understanding the interplay between stability and structural properties, like helicity. Although right-handed G4 structures dominate the experimental data, the molecular basis for this preference over left-handed helicity is unclear. To address this, we employ all-atom molecular dynamics simulations and quantum chemical methods. Our results reveal that right-handed G4s exhibit greater thermodynamic and kinetic stability as a result of favorable sugar-phosphate backbone conformations in guanine tracts. Moreover, while hydrogen-bonding patterns influence helicity-specific G4 loop conformations, they minimally affect stability differences. We also elucidate the strong correlation between helicity and the strand progression direction, essential for G4 structures. These findings deepen our understanding of G4s, providing molecular-level insights into their structural and energetic preferences, which could inform the design of novel nanodevices.

The left-handed Z-conformation of nucleic acids can be adopted by both DNA and RNA when bound by Zα domains found within a variety of viral and innate immune response proteins. While Z-form adoption is preferred by certain sequences, such as the commonly studied (CpG) n repeats, Zα has been reported to bind to a wide range of sequence contexts. Studying how Zα interacts with B-/A-form helices prior to their conversion to the Z-conformation is challenging as binding coincides with Z-form adoption. Here, we studied the binding of Zα from Homo sapiens ADAR1 to a locked "A-type" version of the (CpG) 3 construct (LNA (CpG) 3 ) where the sugar pucker is locked into the C3'- endo /C2'- exo conformation, which prevents the duplex from adopting the alternating C2'/C3'- endo sugar puckers found in the Z-conformation. Using NMR and other biophysical techniques, we find that Zα ADAR1 binds to the LNA (CpG) 3 using a similar interface as for Z-form binding, with a dissociation constant ( K D ) of ∼4 μM. In contrast to Z-DNA/Z-RNA, where two Zα ADAR1 bind to every 6 bp stretch, our data suggests that Zα ADAR1 binds to multiple LNA molecules, indicating a completely different binding mode. Because Zα ADAR1 binds relatively tightly to a non-Z-form model, its binding to B/A-form helices may need to be considered when experiments are carried out which attempt to identify the Z-form targets of Zα domains. The use of LNA constructs may be beneficial in experiments where negative controls for Z-form adoption are needed.

In this study, we designed the 4'- C -acetamidomethyl-2'- O -methoxyethyl (4'- C -ACM-2'- O -MOE) uridine and thymidine modifications, aiming to test them into small interfering RNAs. Thermal melting studies revealed that incorporating a single 4'- C -ACM-2'- O -MOE modification in the DNA duplex reduced thermal stability. In contrast, an increase in thermal stability was observed when the modification was introduced in DNA:RNA hybrid and in siRNAs. Thermal destabilization in DNA duplex was attributed to unfavorable entropy, which was mainly compensated by the enthalpy factor to some extent. A single 4'- C -ACM-2'- O -MOE thymidine modification at the penultimate position of the 3'-end of dT 20 oligonucleotides in the presence of 3'-specific exonucleases, snake venom phosphodiesterase (SVPD), demonstrated significant stability as compared to monomer modifications including 2'- O -Me, 2'- O -MOE, and 2'-F. In gene silencing studies, we found that the 4'- C -ACM-2'- O -MOE uridine or thymidine modifications at the 3'-overhang in the passenger strand in combination with two 2'-F modifications exhibited superior RNAi activity. The results suggest that the dual modification is well tolerated at the 3'-end of the passenger strand, which reflects better siRNA stability and silencing activity. Interestingly, 4'- C -ACM-2'- O -MOE-modified siRNAs showed considerable gene silencing even after 96 h posttransfection; it showed that our modification could induce prolonged gene silencing due to improved metabolic stability. Molecular modeling studies revealed that the introduction of the 4'- C -ACM-2'- O -MOE modification at the 3'-end of the siRNA guide strand helps to anchor the strand within the PAZ domain of the hAgo2 protein. The overall results indicate that the 4'- C -ACM-2'- O -MOE uridine and thymidine modifications are promising modifications to improve the stability, potency, and hAgo2 binding of siRNAs.

Inside cells, proteins complex with nucleic acids to form liquid droplets resulting from liquid-liquid phase separation. The presence of mutated proteins can change the state of these liquid droplets to solids or gels, triggering neurodegenerative diseases. The mechanism of the liquid to solid or gel transition is still unclear. Solutions of poly(l-ornithine- co -l-citrulline) (P L OC) copolymers, which exhibit upper critical solution temperature-type behavior, change state upon cooling. In this study, we evaluated the effect of nucleic acids complexed with P L OC on phase changes. In the presence of nucleic acids, such as polyC and polyU, P L OC formed liquid droplets at low temperatures. The droplets dissolved at temperatures above the phase separation temperature. The phase separation temperature depended on the chemical structure of the nucleobase, implying that electrostatic and hydrogen bonding interactions between the nucleic acid and P L OC influenced phase separation. Furthermore, the liquid droplets spontaneously changed to gel-like precipitates due to spontaneous release of nucleic acids from the complex. The rate of the liquid droplet-to-gel transition depended on the magnitude of electrostatic and hydrogen bonding interactions between P L OC and nucleic acid. P L OC complexed with mRNA also underwent a liquid droplet-to-gel transition upon the release of mRNA. This work provides insights into the mechanism of pathogenic transitions of the cellular droplets.

Loop-mediated isothermal amplification (LAMP) is a rapid, sensitive, and cost-effective method for developing point-of-care nucleic acid testing due to its isothermal nature. Yet, LAMP can suffer from the issue of false positives, which can compromise the specificity of the results. LAMP false positives typically arise due to contamination, nonspecific amplification, and nonspecific signal reporting (intercalating dyes, colorimetric, turbidity, etc.). While dye-labeled primers or probes have been introduced for multiplexed detection and enhanced specificity in LAMP assays, they carry the risk of reaction inhibition. This inhibition can result from the labeled primers with fluorophores or quenchers and probes that do not fully dissociate during reaction. This work demonstrated a nanopore-based system for probe-free LAMP readouts by employing amplicon sizing and counting, analogous to an electronic version of gel electrophoresis. We first developed a model to explore LAMP kinetics and verified distinct patterns between true and false positives via gel electrophoresis. Subsequently, we implemented nanopore sized counting and calibrated the event charge deficit (ECD) values and frequencies to ensure a fair analysis of amplicon profiles. This sized counting method, integrated with machine learning, achieved 91.67% accuracy for false positive discrimination, enhancing LAMP's reliability for nucleic acid detection.

CRISPR/Cas12a-based biosensing is advancing rapidly; however, achieving sensitive and cost-effective reporting of Cas12a activation remains a challenge. In response, we have developed a label-free system capable of postamplifying Cas12a activation by integrating hybridization chain reaction (HCR) and DNA-copper nanoclusters (DNA-CuNCs). The trans-cleavage of Cas12a triggers a silenced HCR, leading to the in situ assembly of fluorescent DNA-CuNCs, allowing for the turn-on reporting of Cas12a activation. Without preamplification, this assay can detect DNA with a detection limit of 5 fM. Furthermore, when coupled with preamplification, the system achieves exceptional sensitivity, detecting the monkeypox virus (MPXV) plasmid at 1 copy in human serum. In a MPXV pseudovirus-based validation test, the obtained results are in agreement with those obtained by qPCR, reinforcing the robustness of this method. Our study represents the first effort to manipulate DNA-CuNC formation on HCR for highly sensitive and cost-effective reporting of Cas12a, resulting in an efficient synthetic biology-enabled sensing platform for biosafety applications.

Spherical nucleic acids (SNAs) have been used to construct various nanobiosensors with gold nanoparticles (AuNPs) as nuclei. The SNAs play a critical role in biosensing due to their various physical and chemical properties, programmability, and specificity recognition ability. In this study, CRISPR-responsive self-assembled spherical nucleic acid (CRISPR-rsSNA) detection probes were constructed by conjugating fluorescein-labeled probes to the surface of AuNPs to improve the sensing performance. Also, the mechanism of ssDNA and the role of different fluorescent groups in the self-assembly process of CRISPR-rsSNA were explored. Then, CRISPR-rsSNA and reverse transcription-recombinase polymerase amplification (RT-RPA) were combined to develop an ultrasensitive fluorescence-detection strategy for norovirus. In the presence of the virus, the target RNA sequence of the virus was transformed and amplified by RT-RPA. The resulting dsDNA activated the trans-cleavage activity of CRISPR cas12a, resulting in disintegrating the outer nucleic acid structure of the CRISPR-rsSNA at a diffusible rate, which released reporter molecules. Norovirus was quantitated by fluorescence detection. This strategy facilitated the detection of the norovirus at the attomolar level. An RT-RPA kit for norovirus detected would be developed based on this method. The proposed method would be used for the detection of different viruses just by changing the target RNA and crRNA of the CRISPR cas12a system which provided a foundation for high-throughput detection of various substances.

Nucleic acid analysis with an easy-to-use workflow, high specificity and sensitivity, independence of sophisticated instruments, and accessibility outside of the laboratory is highly desirable for the detection and monitoring of infectious diseases. Integration of laboratory-quality sample preparation on a hand-held system is critical for performance. A SlipChip device inspired by the combination lock can perform magnetic bead-based nucleic acid extraction with several clockwise and counterclockwise rotations. A palm-sized base station was developed to assist sample preparation and provide thermal control of isothermal nucleic acid amplification without plug-in power. The loop-mediated isothermal amplification reaction can be performed with a colorimetric method and directly analyzed by the naked eye or with a mobile phone app. This system achieves good bead recovery during the sample preparation workflow and has minimal residue carryover from the lysis and elution buffers. Its performance is comparable to that of the standard laboratory protocol with real-time qPCR amplification methods. The entire workflow is completed in less than 35 min and the device can achieve 500 copies/mL sensitivity. Thirty clinical nasal swab samples were collected and tested with a sensitivity of 95% and a specificity of 100% for SARS-CoV-2. This combination-lock SlipChip provides a promising fast, easy-to-use nucleic acid test with bead-based sample preparation that produces laboratory-quality results for point-of-care settings, especially in home use applications.

Cell-free protein synthesis (CFPS) has recently gained considerable attention as a new platform for developing methods to detect various molecules, ranging from small chemicals to biological macromolecules. Retroreflection has been used as an alternative signal to develop analytical methods because it can be detected by using a simple instrument comprising a white light source and a camera. Here, we report a novel reporter protein that couples the capability of CFPS and the simplicity of retroreflection signal detection. The design of the reporter was based on two pairs of protein-peptide interactions, SpyCatcher003-SpyTag003 and MDM2-PMI(N8A). MDM2-MDM2-SpyCatcher003 was decided as the reporter protein, and the two peptides, SpyTag003 and PMI(N8A), were immobilized on the surfaces of retroreflective Janus particles and microfluidic chips, respectively. The developed retroreflection signal detection system was combined with a previously reported CFPS reaction that can transduce the presence of a single-stranded nucleic acid into protein synthesis. The resulting methods were applied to detect 16S rRNAs of several foodborne pathogens. Concentration-dependent relationships were observed over a range of 10° fM to 10 2 pM, with the limits of detection being single-digit femtomolar concentrations. Considering the designability of the CFPS system for other targets, the retroreflection signal detection method will enable the development of novel methods to detect various molecules.

DNA-based devices such as DNA logic gates self-assemble into supramolecular structures, as dictated by the sequences of the constituent oligonucleotides and their predictable Watson-Crick base pairing interactions. The programmable nature of DNA-based devices permits the design and implementation of DNA circuits that interact in a dynamic and sequential manner capable of spatially arranging disparate DNA species. Here, we report the application of an activatable fluorescence reporter based on a proximity-driven inverse electron demand Diels-Alder (IEDDA) reaction and its robust integration with DNA strand displacement circuits. In response to specific DNA input patterns, sequential strand displacement reactions are initiated and culminate in the hybridization of two modified DNA strands carrying probes capable of undergoing an IEDDA reaction between a vinyl-ether-caged fluorophore and its reactive partner tetrazine, leading to the activation of fluorescence. This approach provides a major advantage for DNA computing in mammalian cells since circuit degradation does not induce fluorescence, in contrast to traditional fluorophore-quencher designs. We demonstrate the robustness and sensitivity of the reporter by testing its ability to serve as a readout for DNA logic circuits of varying complexity inside cells.

Specific and sensitive detection of microRNAs continues to encounter significant challenges, especially in the development of rapid and efficient isothermal amplification strategies for point-of-care settings. The exponential amplification reaction (EXPAR) has garnered significant attention owing to its simplicity and rapid amplification of signals within a short period. However, a substantial loss of amplification efficiency, difficulty in distinguishing closely related homologous sequences, and adapting the designed templates to other targets seriously hamper the practical application of the EXPAR. In this work, a hairpin template tailored for the EXPAR system (exp-Hairpin) was constructed by adding identical trigger sequences and enzyme cleavage sites on two arms of the hairpin, achieving theoretically more than 2 n amplification efficiency and minimal background amplification of EXPAR. Modulating the stability of the exp-Hairpin template by increasing the stem length, the specificity of detecting target miRNA in highly homologous sequences could be significantly improved. Using miRNA let-7a as a target model, the exp-Hairpin with 8 bp stem length for EXPAR amplification curves could effectively distinguish target let-7a and nontarget let-7b/7c/7f/7g/7i homologous sequences. This strategy enabled the sensitive and accurate analysis of let-7a in diluted human serum with satisfactory recoveries. By simply replacing the loop recognition sequence of exp-Hairpin, the specific detection of miR-200b was also achieved, demonstrating the universality of this strategy. The exp-Hairpin EXPAR accelerates simple and rapid molecular diagnostic applications for short nucleic acids.

In recent years, the CRISPR/Cas12a-based sensing strategy has shown significant potential for specific target detection due to its rapid and sensitive characteristics. However, the "always active" biosensors are often insufficient to manipulate nucleic acid sensing with high spatiotemporal control. It remains crucial to develop nucleic acid sensing devices that can be activated at the desired time and space by a remotely applied stimulus. Here, we integrated photoactivation with the CRISPR/Cas12a system for DNA and RNA detection, aiming to provide high spatiotemporal control for nucleic acid sensing. By rationally designing the target recognition sequence, this photoactivation CRISPR/Cas12a system could recognize HPV16 and survivin , respectively. We combined the lateral flow assay strip test with the CRISPR/Cas12a system to realize the visualization of nucleic acid cleavage signals, displaying potential instant test application capabilities. Additionally, we also successfully realized the temporary control of its fluorescent sensing activity for survivin by photoactivation in vivo , allowing rapid detection of target nucleic acids and avoiding the risk of contamination from premature leaks during storage. Our strategy suggests that the CRISPR/Cas12a platform can be triggered by photoactivation to sense various targets, expanding the technical toolbox for precise biological and medical analysis. This study represents a significant advancement in nucleic acid sensing and has potential applications in disease diagnosis and treatment.

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.

Endonuclease V (EndoV) is a single-metal-dependent enzyme that repairs deaminated DNA nucleobases in cells by cleaving the phosphodiester bond, and this enzyme has proven to be a powerful tool in biotechnology and medicine. The catalytic mechanism used by EndoV must be understood to design new disease detection and therapeutic solutions and further exploit the enzyme in interdisciplinary applications. This study has used a mixed molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) approach to compare eight distinct catalytic pathways and provides the first proposed mechanism for bacterial EndoV. The calculations demonstrate that mechanisms involving either direct or indirect metal coordination to the leaving group of the substrate previously proposed for other nucleases are unlikely for EndoV, regardless of the general base (histidine, aspartate, and substrate phosphate moiety). Instead, distinct catalytic pathways are characterized for EndoV that involve K139 stabilizing the leaving group, a metal-coordinated water stabilizing the transition structure, and either H214 or a substrate phosphate group activating the water nucleophile. In silico K139A and H214A mutational results support the newly proposed roles of these residues. Although this is a previously unseen combination of general base, general acid, and metal-binding architecture for a one-metal-dependent endonuclease, our proposed catalytic mechanisms are fully consistent with experimental kinetic, structural, and mutational data. In addition to substantiating a growing body of literature, suggesting that one metal is enough to catalyze P-O bond cleavage in nucleic acids, this new fundamental understanding of the catalytic function will promote the exploration of new and improved applications of EndoV.

Protein-molecule interactions play a crucial role in various biological functions, with their accurate prediction being pivotal for drug discovery and design processes. Traditional methods for predicting protein-molecule interactions are limited. Some can only predict interactions with a specific molecule, restricting their applicability, while others target multiple molecule types but fail to efficiently process diverse interaction information, leading to complexity and inefficiency. This study presents a novel deep learning model, MucLiPred, equipped with a dual contrastive learning mechanism aimed at improving the prediction of multiple molecule-protein interactions and the identification of potential molecule-binding residues. The residue-level paradigm focuses on differentiating binding from non-binding residues, illuminating detailed local interactions. The type-level paradigm, meanwhile, analyzes overarching contexts of molecule types, like DNA or RNA, ensuring that representations of identical molecule types gravitate closer in the representational space, bolstering the model's proficiency in discerning interaction motifs. This dual approach enables comprehensive multi-molecule predictions, elucidating the relationships among different molecule types and strengthening precise protein-molecule interaction predictions. Empirical evidence demonstrates MucLiPred's superiority over existing models in robustness and prediction accuracy. The integration of dual contrastive learning techniques amplifies its capability to detect potential molecule-binding residues with precision. Further optimization, separating representational and classification tasks, has markedly improved its performance. MucLiPred thus represents a significant advancement in protein-molecule interaction prediction, setting a new precedent for future research in this field.

Rapid and accurate diagnosis is crucial for managing the global health threat posed by multidrug-resistant bacterial infections; however, current methods have limitations in either being time-consuming, labor-intensive, or requiring instruments with high costs. Addressing these challenges, we introduce a wireless electrochemical sensor integrating the CRISPR/Cas system with electroconductive polymer dot (PD) nanoparticles to rapidly detect bacterial pathogens from human sputum. To enhance the electroconductive properties, we synthesized copper-ion-immobilized PD (PD-Cu), followed by conjugation of the deactivated Cas9 protein (dCas9) onto PD-Cu-coated Si electrodes to generate the dCas9-PD-Cu sensor. The dCas9-PD-Cu sensor integrated with isothermal amplification can specifically detect target nucleic acids of multidrug-resistant bacteria, such as the antibiotic resistance genes kpc-2 and mecA . The dCas9-PD-Cu sensor exhibits high sensitivity, allowing for the detection of ∼54 femtograms of target nucleic acids, based on measuring the changes in resistivity of the Si electrodes through target capture by dCas9. Furthermore, a wireless sensing platform of the dCas9-PD-Cu sensor was established using a Bluetooth module and a microcontroller unit for detection using a smartphone. We demonstrate the feasibility of the platform in diagnosing multidrug-resistant bacterial pneumonia in patients' sputum samples, achieving 92% accuracy. The current study presents a versatile biosensor platform that can overcome the limitations of conventional diagnostics in the clinic.

We report a thorough investigation of the role of single-stranded thymidine (ssT) linkers in the stability and flexibility of minimal, multistranded DNA nanostructures. We systematically explore the impact of varying the number of ssTs in three-way junction motifs (3WJs) on their formation and properties. Through various UV melting experiments and molecular dynamics simulations, we demonstrate that while the number of ssTs minimally affects thermodynamic stability, the increasing ssT regions significantly enhance the structural flexibility of 3WJs. Utilizing this knowledge, we design triangular DNA nanoparticles with varying ssTs, all showing exceptional assembly efficiency except for the 0T triangle. All triangles demonstrate enhanced stability in blood serum and are nonimmunostimulatory and nontoxic in mammalian cell lines. The 4T 3WJ is chosen as the building block for constructing other polygons due to its enhanced flexibility and favorable physicochemical characteristics, making it a versatile choice for creating cost-effective, stable, and functional DNA nanostructures that can be stored in the dehydrated forms while retaining their structures. Our study provides valuable insights into the design and application of nucleic acid nanostructures, emphasizing the importance of understanding stability and flexibility in the realm of nucleic acid nanotechnology. Our findings suggest the intricate connection between these ssTs and the structural adaptability of DNA 3WJs, paving the way for more precise design and engineering of nucleic acid nanosystems suitable for broad biomedical applications.

Detection of nucleic acids from a single multiplexed and amplification-free test is critical for ensuring food safety, clinical diagnostics, and environmental monitoring. In this study, we introduced a mesophilic Argonaute protein from Clostridium butyricum (CbAgo), which exhibits nucleic acid endonuclease activity, to achieve a programmable, amplification-free system (PASS) for rapid nucleic acid quantification at ambient temperatures in one pot. By using CbAgo-mediated binding with specific guide DNA (gDNA) and subsequent targeted cleavage of wild-type target DNAs complementary to gDNA, PASS can detect multiple foodborne pathogen DNA (<10 2 CFU/mL) simultaneously. The fluorescence signals were then transferred to polydisperse emulsions and analyzed by using deep learning. This simplifies the process and increases the suitability of polydisperse emulsions compared to traditional digital PCR, which requires homogeneous droplets for accurate detection. We believe that PASS has the potential to become a next-generation point-of-care digital nucleic acid detection method.

Intracellular bacterial infections bring a considerable risk to human life and health due to their capability to elude immune defenses and exhibit significant drug resistance. As a result, confronting and managing these infections present substantial challenges. In this study, we developed a multifunctional living phage nanoconjugate by integrating aggregation-induced emission luminogen (AIEgen) photosensitizers and nucleic acids onto a bacteriophage framework (forming MS2-DNA-AIEgen bioconjugates). These nanoconjugates can rapidly penetrate mammalian cells and specifically identify intracellular bacteria while concurrently producing a detectable fluorescent signal. By harnessing the photodynamic property of AIEgen photosensitizer and the bacteriophage's inherent targeting and lysis capability, the intracellular bacteria can be effectively eliminated and the activity of the infected cells can be restored. Moreover, our engineered phage nanoconjugates were able to expedite the healing process in bacterially infected wounds observed in diabetic mice models while simultaneously enhancing immune activity within infected cells and in vivo , without displaying noticeable toxicity. We envision that these multifunctional phage nanoconjugates, which utilize AIEgen photosensitizers and spherical nucleic acids, may present a groundbreaking strategy for combating intracellular bacteria and offer powerful avenues for theranostic applications in intracellular bacterial infection-associated diseases.

Strategies for rapid, effective nucleic acid processing hold tremendous significance to the clinical analysis of circulating tumor DNA ( ctDNA ), a family of important markers indicating tumorigenesis and metastasis. However, traditional techniques remain challenging to achieve efficient DNA enrichment, further bringing about complicated operation and limited detection sensitivity. Here, we developed an ion concentration polarization microplatform that enabled highly rapid, efficient enrichment and purification of ctDNA from a variety of clinical samples, including serum, urine, and feces. The platform demonstrated efficiently separating and enriching ctDNA within 30 s, with a 100-fold improvement over traditional methods. Integrating an on-chip isothermal amplification module, the platform further achieved 100-fold enhanced sensitivity in ctDNA detection, which significantly eliminated false-negative results in the serum or urine samples due to the low abundance of ctDNA . Such a simple-designed platform offers a user-friendly yet powerful diagnosis technique with a wide applicability, ranging from early tumor diagnosis to infection screening.

Silver cations can mediate base pairing of guanine (G) DNA oligomers, yielding linear parallel G-Ag + -G duplexes with enhanced stabilities compared to those of canonical DNA duplexes. To enable their use in programmable DNA nanotechnologies, it is critical to understand solution-state formation and the nanomechanical stiffness of G-Ag + -G duplexes. Using temperature-controlled circular dichroism (CD) spectroscopy, we find that heating mixtures of G oligomers and silver salt above 50 °C fully destabilizes G-quadruplex structures and converts oligomers to G-Ag + -G duplexes. Electrospray ionization mass spectrometry supports that G-Ag + -G duplexes form at stoichiometries of 1 Ag + per base pair, and CD spectroscopy suggests that as the Ag + /base stoichiometry increases further, G-Ag + -G duplexes undergo additional morphological changes. Using liquid-phase atomic force microscopy, we find that this excess Ag + enables assembly of long fiberlike structures with ∼2.5 nm heights equivalent to a single DNA duplex but with lengths that far exceed a single duplex. Finally, using the conditions established to form single G-Ag + -G duplexes, we use a surface forces apparatus (SFA) to compare the solution-phase stiffness of single G-Ag + -G duplexes with dG-dC Watson-Crick-Franklin duplexes. SFA shows that G-Ag + -G duplexes are 1.3 times stiffer than dG-dC duplexes, confirming gas-phase ion mobility spectrometry measurements and computational predictions. These findings may guide the development of structural DNA nanotechnologies that rely on silver-mediated base pairing.

The left-handed Z-conformation of nucleic acids can be adopted by both DNA and RNA when bound by Zα domains found within a variety of innate immune response proteins. Zα domains stabilize this higher-energy conformation by making specific interactions with the unique geometry of Z-DNA/Z-RNA. However, the mechanism by which a right-handed helix contorts to become left-handed in the presence of proteins, including the intermediate steps involved, is poorly understood. Through a combination of nuclear magnetic resonance (NMR) and other biophysical measurements, we have determined that in the absence of Zα, under low salt conditions at room temperature, d(CpG) and r(CpG) constructs show no observable evidence of transient Z-conformations greater than 0.5% on either the intermediate or slow NMR time scales. At higher temperatures, we observed a transient unfolded intermediate. The ease of melting a nucleic acid duplex correlates with Z-form adoption rates in the presence of Zα. The largest contributing factor to the activation energies of Z-form adoption as calculated by Arrhenius plots is the ease of flipping the sugar pucker, as required for Z-DNA and Z-RNA. Together, these data validate the previously proposed "zipper model" for Z-form adoption in the presence of Zα. Overall, Z-conformations are more likely to be adopted by double-stranded DNA and RNA regions flanked by less stable regions and by RNAs experiencing torsional/mechanical stress.

Exposure to the physicochemical agents that interact with nucleic acids (NA) may lead to modification of DNA and RNA (i.e., NA modifications), which have been associated with various diseases, including cancer. The emerging field of NA adductomics aims to identify both known and unknown NA modifications, some of which may also be associated with proteins. One of the main challenges for adductomics is the processing of massive and complex data generated by high-resolution tandem mass spectrometry (HR-MS/MS). To address this, we have developed a software called " FeatureHunter ", which provides the automated extraction, annotation, and classification of different types of key NA modifications based on the MS and MS/MS spectra acquired by HR-MS/MS, using a user-defined feature list. The capability and effectiveness of FeatureHunter was demonstrated by analyzing various NA modifications induced by formaldehyde or chlorambucil in mixtures of calf thymus DNA, yeast RNA and proteins, and by analyzing the NA modifications present in the pooled urines of smokers and nonsmokers. The incorporation of FeatureHunter into the NA adductomics workflow offers a powerful tool for the identification and classification of various types of NA modifications induced by reactive chemicals in complex biological samples, providing a valuable resource for studying the exposome.

DNA origami is a popular nanofabrication strategy that employs self-assembly of a long single scaffold strand, typically less than 10 kilobases in length, with hundreds of shorter staple strands into a desired shape. In particular, origami arranged as a single-layer rectangle has proven popular as flat pegboards that can display functionalities at staple-strand breakpoints, off the sides of the constituent double helices, with a ∼5.3 nm rhombic-lattice spacing. For applications that demand tighter spacing, functionalities can be displayed instead on the termini of helices of multilayer DNA origami. However, pegboards with the greatest addressable surface area are often found to be the most versatile. Given the practical limitations of the length of the scaffold that can be easily realized, designs that minimize the length of each helix would have advantages for maximizing the number of helices and therefore the number of addressable pixels on each terminal surface. Here we present an architecture for multilayer DNA origami displaying flush terminal interfaces from over 200 helices that each are only 5.3 turns in length. We characterize an example using cryo-EM imaging paired with single-particle analysis for further analysis of the global structure.

The COVID-19 pandemic proved the great usefulness of lateral flow tests as self- and rapid tests. The rapid expansion of this field requires the design and validation of novel, affordable, and versatile technologies for the easy fabrication of a variety of lateral flow devices. In the present work, we have developed a new, simple, and cost-effective system for the dispensing of reagents on the membranes of lateral flow devices to be used for research purposes. The 3D printing technology is integrated, for the first time, with simple and inexpensive tools such as a technical pen and disposable pipet tips for the construction of the test and the control areas of the devices. We also used this system for the automated fabrication of spots on the membrane for multiplex analysis. The devices were applied for the detection of proteins/antibodies and single- and double-stranded DNA targets. Also, devices with multiple biosensing areas on the membrane were constructed for the simultaneous detection of different analytes. The proposed system is very simple, automated, and inexpensive and has provided rapid and reproducible construction of lateral flow devices. Compared to a commercially available automated dispenser, the devices showed similar detection capabilities and reproducibility in various real samples. Moreover, contrary to the existing dispensers, the proposed system does not require any gas or costly precision pumps and syringes for the deposition. In conclusion, the developed 3D printer-based system could be an extremely useful alternative for research laboratories for the construction of lateral flow devices of various assay configurations.

Ion mobility spectrometry (IMS) has become popular to characterize biomolecule folding. Numerous studies have shown that proteins that are folded in solution remain folded in the gas phase, whereas proteins that are unfolded in solution adopt more extended conformations in the gas phase. Here, we discuss how general this tenet is. We studied single-stranded DNAs (human telomeric cytosine-rich sequences with CCCTAA repeats), which fold into an intercalated motif (i-motif) structure in a pH-dependent manner, thanks to the formation of C–H+–C base pairs. As i-motif formation is favored at low ionic strength, we could investigate the ESI-IMS-MS behavior of i-motif structures at pH ~ 5.5 over a wide range of ammonium acetate concentrations (15 to 100 mM). The control experiments consisted of either the same sequence at pH ~ 7.5, wherein the sequence is unfolded, or sequence variants that cannot form i-motifs (CTCTAA repeats). The surprising results came from the control experiments. We found that the ionic strength of the solution had a greater effect on the compactness of the gas-phase structures than the solution folding state. This means that electrosprayed ions keep a memory of the charging process, which is influenced by the electrolyte concentration. We discuss these results in light of the analyte partitioning between the droplet interior and the droplet surface, which in turn influences the probability of being ionized via a charged residue-type pathway or a chain extrusion-type pathway. [ABSTRACT FROM AUTHOR]

Ribonucleic acid (RNA)-based therapies have become an attractive topic in disease intervention, especially with some that have been approved by the FDA such as the mRNA COVID-19 vaccine (Comirnaty, Pfizer-BioNTech, and Spikevax, Moderna) and Patisiran (siRNA-based drug for liver delivery). However, extensive applications are still facing challenges in delivering highly negatively charged RNA to the targeted site. Therapeutic delivery strategies including RNA modifications, RNA conjugates, and RNA polyplexes and delivery platforms such as viral vectors, nanoparticle-based delivery platforms, and hydrogel-based delivery platforms as potential nucleic acid-releasing depots have been developed to enhance their cellular uptake and protect nucleic acid from being degraded by immune systems. Here, we review the growing number of viral vectors, nanoparticles, and hydrogel-based RNA delivery systems; describe RNA loading/release mechanism induced by environmental stimulations including light, heat, pH, or enzyme; discuss their physical or chemical interactions; and summarize the RNA therapeutics release period (temporal) and their target cells/organs (spatial). Finally, we describe current concerns, highlight current challenges and future perspectives of RNA-based delivery systems, and provide some possible research areas that provide opportunities for clinical translation of RNA delivery carriers.

CRISPR/Cas system is becoming an increasingly influential technology that has been repositioned in nucleic acid detection. A preamplification step is usually required to improve the sensitivity of CRISPR/Cas-based detection. The striking biological features of CRISPR/Cas, including programmability, high sensitivity and sequence specificity, and single-base resolution. More strikingly, the target-activated trans -cleavage could act as a biocatalytic signal transductor and amplifier, thereby empowering it to potentially perform nucleic acid detection without a preamplification step. The reports of such work are on the rise, which is not only scientifically significant but also promising for futuristic end-user applications. This review started with the introduction of the detection methods of nucleic acids and the CRISPR/Cas-based diagnostics (CRISPR-Dx). Next, we objectively discussed the pros and cons of preamplification steps for CRISPR-Dx. We then illustrated and highlighted the recently developed strategies for CRISPR/Cas-powered amplification-free detection that can be realized through the uses of ultralocalized reactors, cascade reactions, ultrasensitive detection systems, or others. Lastly, the challenges and futuristic perspectives were proposed. It can be expected that this work not only makes the researchers better understand the current strategies for this emerging field, but also provides insight for designing novel CRISPR-Dx without a preamplification step to win practicable use in the near future.

The interaction of small molecules or proteins with RNA or DNA often involves changes in the nucleic acid (NA) folding and structure. A biophysical characterization of these processes helps us to understand the underlying molecular mechanisms. Here, we propose kinFRET (kinetics Förster resonance energy transfer), a real-time ensemble FRET methodology to measure binding and folding kinetics. With kinFRET, the kinetics of conformational changes of NAs (DNA or RNA) upon analyte binding can be directly followed via a FRET signal using a chip-based biosensor. We demonstrate the utility of this approach with two representative examples. First, we monitored the conformational changes of different formats of an aptamer (MN19) upon interaction with small-molecule analytes. Second, we characterized the binding kinetics of RNA recognition by tandem K homology (KH) domains of the human insulin-like growth factor II mRNA-binding protein 3 (IMP3), which reveals distinct kinetic contributions of the two KH domains. Our data demonstrate that kinFRET is well suited to study the kinetics and conformational changes of NA-analyte interactions.

Mg 2+ ions play an essential part in stabilizing the tertiary structure of nucleic acids. While the importance of these ions is well documented, their localization and elucidation of their role in the structure and dynamics of nucleic acids are often challenging. In this work, pulsed electron-electron double resonance spectroscopy (PELDOR, also known as DEER) was used to localize two high affinity divalent metal ion binding sites in the tetracycline RNA aptamer with high accuracy. For this purpose, the aptamer was labeled at different positions with a semirigid nitroxide spin label and diamagnetic Mg 2+ was replaced with paramagnetic Mn 2+ , which did not alter the folding process or ligand binding. Out of the several divalent metal ion binding sites that are known from the crystal structure, two binding sites with high affinity were detected: one that is located at the ligand binding center and another at the J1/2 junction of the RNA.