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A novel photoelectrochemical (PEC) biosensor was developed incorporating a specifically designed RNA aptamer for the detection of theophylline (TP). This involved utilizing two nucleotide base aptamers with tailored sequences designed to target TP. The 3' end of a single-stranded RNA sequence (5'-GGAUACCA-(CH2)6-SH-3') and the 5' end of a complementary stranded RNA sequence (5'-HS-(CH2)6-CCUUGGAAGCC-3') were linked to gold nanoparticles (AuNPs) and CdS quantum dots (QDs), respectively. These two single-stranded RNAs (ssRNA) formed a double-stranded RNA (dsRNA) capable of recognizing TP. This major structural change altered the spacing between QDs and NPs, which signaled the presence and concentration of TP. TP was photoelectrochemical catalytic oxidation by the hole of CdS QDs under illumination, then anode photocurrent was generated. Due to the increase in surface impedance and the effect of exciton energy transfer (EET) between QDs and AuNPs, the photocurrent would undergo varying degrees of change. TP was detected by changes in photocurrent. PEC detection of TP was achieved in the range of 0.1 µM-200 µM. The detection limit was 0.033 µM. The method exhibited commendable reproducibility and remarkable selectivity. The biosensor was used to measure TP content in tea, beverages and blood samples, resulting in satisfactory recovery rates.

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The human transmembrane protein Transferrin Receptor-1 is regarded as a promising target for the systemic delivery of therapeutic agents, particularly of nucleic acid therapeutics, such as short double stranded RNAs. This ubiquitous receptor is involved in cellular iron uptake, keeping intracellular homeostasis. It is overexpressed in multiple cancer cell types and is internalized via clathrin-mediated endocytosis. In previous studies, a human transferrin receptor-1 RNA aptamer, identified as TR14 ST1-3, was shown to be capable of effectively internalizing into cells in culture and to deliver small, double stranded RNAs in vitro and in vivo, via systemic administration. To understand, at the molecular level, the aptamer binding to the receptor and the impact of conjugation with the therapeutic RNA, a multi-level in silico protocol was employed, including protein-aptamer docking, molecular dynamics simulations and free energy calculations. The competition for the binding pocket, between the aptamer and the natural ligand human Transferrin, was also evaluated. The results show that the aptamer binds to the same region as Transferrin, with residues from the helical domain showing a critical role. Moreover, the conjugation to the therapeutic RNA, was shown not to affect aptamer binding. Overall, this study provides an atomic-level understanding of aptamer association to human Transferrin Receptor-1 and of its conjugation with a short model-therapeutic RNA, providing also important clues for futures studies aiming to deliver other oligonucleotide-based therapeutics via Transferrin Receptor.

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Fluorogenic RNA aptamers, which specifically bind to fluorogens and dramatically enhance their fluorescence, are valuable for imaging and detecting RNAs and metabolites in living cells. Most fluorogenic RNA aptamers have been identified and engineered through iterative rounds of in vitro selection based on their binding to target fluorogens. While such selection is an efficient approach for generating RNA aptamers, it is less efficient for isolating fluorogenic aptamers because it does not directly screen for fluorogenic properties. In this study, we combined a fluorescence-based in vitro selection technique using water-in-oil microdroplets with an affinity-based selection technique to obtain fluorogenic RNA aptamers. This approach allowed us to identify novel fluorogenic aptamers for a biotin-modified thiazole orange derivative. Our results demonstrate that our approach can expand the diversity of fluorogenic RNA aptamers, thus leading to new applications for the imaging and detection of biomolecules.

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Hemostasis relies on a reaction network of serine proteases and their cofactors to form a blood clot. Coagulation factor IXa (protease) plays an essential role in hemostasis as evident from the bleeding disease associated with its absence. RNA aptamers specifically targeting individual coagulation factors have potential as anticoagulants and as probes of the relationship between structure and function. Here, we report X-ray structures of human factor IXa without a ligand bound to the active site either in the apo-form or in complex with an inhibitory aptamer specific for factor IXa. The aptamer binds to an exosite in the catalytic domain and allosterically distorts the active site. Our studies reveal a conformational ensemble of IXa states, wherein large movements of Trp215 near the active site drive functional transitions between the closed (aptamer-bound), latent (apo), and open (substrate-bound) states. The latent state of the apo-enzyme may bear on the uniquely poor catalytic activity of IXa compared to other coagulation proteases. The exosite, to which the aptamer binds, has been implicated in binding VIIIa and heparin, both of which regulate IXa function. Our findings reveal the importance of exosite-driven allosteric modulation of IXa function and new strategies to rebalance hemostasis for therapeutic gain.

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Aptamers are nucleic acid sequences that specifically bind with target molecules and are vital to applications such as biosensing, drug development, disease diagnostics, etc. The traditional selection procedure of aptamers is based on the Systematic Evolution of Ligands by an Exponential Enrichment (SELEX) process, which relies on repeating cycles of screening and amplification. With the rapid development of aptamer applications, RNA and XNA aptamers draw more attention than before. But their selection is troublesome due to the necessary reverse transcription and transcription process (RNA) or low efficiency and accuracy of enzymes for amplification (XNA). In light of this, we review the recent advances in aptamer selection methods and give an outlook on future development in a non-SELEX approach, which simplifies the procedure and reduces the experimental costs. We first provide an overview of the traditional SELEX methods mostly designed for screening DNA aptamers to introduce the common tools and methods. Then a section on the current screening methods for RNA and XNA is prepared to demonstrate the efforts put into screening these aptamers and the current difficulties. We further predict that the future trend of aptamer selection lies in non-SELEX methods that do not require nucleic acid amplification. We divide non-SELEX methods into an immobilized format and non-immobilized format and discuss how high-resolution partitioning methods could facilitate the further improvement of selection efficiency and accuracy.

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Bacillus anthracis, the causative agent of anthrax, poses a substantial threat to public health and national security, and is recognized as a potential bioweapon due to its capacity to form resilient spores with enduring viability. Inhalation or ingestion of even minute quantities of aerosolized spores can lead to widespread illness and fatalities, underscoring the formidable lethality of the bacterium. With an untreated mortality rate of 100%, Bacillus anthracis is a disconcerting candidate for bioterrorism. In response to this critical scenario, we employed state-of-the-art computational tools to conceive and characterize flexible RNA aptamer therapeutics tailored for anthrax. The foundational structure of the flexible RNA aptamers was designed by removing the C2'-C3' in each nucleotide unit. Leveraging the crystal structure of Bacillus anthracis ribosomal protein S8 complexed with an RNA aptamer, we explored the structural, dynamic, and energetic aspects of the modified RNA aptamer - S8 protein complexes through extensive all-atom explicit-solvent molecular dynamics simulations (400 ns, 3 replicas each), followed by drawing comparisons to the control system. Our findings demonstrate the enhanced binding competencies of the flexible RNA aptamers to the S8 protein via better shape complementarity and improved H-bond network compared to the control RNA aptamer. This research offers valuable insights into the development of RNA aptamer therapeutics targeting Bacillus anthracis, paving the way for innovative strategies to mitigate the impact of this formidable pathogen.

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In this paper, one of the great challenges faced by silicon-based biosensors is resolved using a biomaterial multilayer. Tiny biomolecules are deposited on silicon substrates, producing devices that have the ability to act as iridescent color sensors. The color is formed by a coating of uniform microstructures through the interference of light. The system exploits a flat, RNA-aptamer-coated silicon-based surface to which captured microbes are covalently attached. Silicon surfaces are encompassed with the layer-by-layer deposition of biomolecules, as characterized by atomic force microscopy and X-ray photoelectron spectroscopy. Furthermore, the results demonstrate an application of an RNA aptamer chip for sensing a specific bacterium. Interestingly, the detection limit for the microbe was observed to be 2 × 106 CFUmL-1 by visually observed color changes, which were confirmed further using UV-Vis reflectance spectrophotometry. In this report, a flexible method has been developed for the detection of the pathogen Sphingobium yanoikuyae, which is found in non-beverage alcohols. The optimized system is capable of detecting the specific target microbe. The simple concept of these iridescent color changes is mainly derived from the increase in thickness of the nano-ordered layers.

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Accurate and sensitive monitoring of the concentration change of anti-digoxigenin (Anti-Dig) antibody is of great importance for diagnosing infectious and immunological diseases. Combining a novel triplex aptamer nanoswitch and the high signal-to-noise ratio of lighting-up RNA aptamer signal amplification, a label-free and ultrasensitive fluorescent sensing approach for detecting Anti-Dig antibodies is described. The target Anti-Dig antibodies recognize and bind with the nanoswitch to open its triplex helix stem structure to release Taq DNA polymerase and short ssDNA primer simultaneously, which activates the Taq DNA polymerase to initiate downstream strand extension of ssDNA primer to yield specific dsDNA containing RNA promoter sequence. T7 RNA polymerase recognizes and binds to these promoter sequences to initiate RNA transcription reaction to produce many RNA aptamer sequences. These aptamers can recognize and bind with Malachite Green (MG) dye specifically and produce highly amplified fluorescent signal for monitoring Anti-Dig antibodies from 50 pM to 50 nM with a detection limit down to 33 pM. The method also exhibits high selectivity for Anti-Dig antibodies and can be used to discriminate trace Anti-Dig antibodies in diluted serum samples. Our method is superior to many immunization-based Anti-Dig antibody detection methods and thus holds great potential for monitoring disease progression and efficacy.

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Aptamers are a class of molecular recognition elements that exhibit high binding affinity and specificity against their respective targets. In view of the many advantages aptamers harbor over their counterpart antibodies, we were impelled to isolate an RNA aptamer against progesterone receptor, particularly its DNA binding domain. A total of eight SELEX cycles were executed against the recombinant Progesterone Receptor DNA-binding domain (PR DBD). The RNA-protein complex in the gel shift assay was subjected to crush and soak method to elute the binders prior to conventional sequencing, the step of which was based upon to coin the term CRUSOAK-SELEX. The sequencing revealed three different classes of sequences, with one class termed, PRapt-3, showing the strongest binding against PR DBD. The dissociation constant of PRapt-3 RNA aptamer was estimated at 380 nM ± 35 nM. PRapt-3 was successfully used to develop aptamer-based diagnostic assays such as ELASA, aptamer-based dot blot, and aptamer-based western blot. The prominent highlight is the performance of the aptamer in aptacytostaining, which was unachievable with antibodies. Compared to its counterpart antibodies, PRapt-3 has a better penetration capacity in aptahistostaining using the formalin-fixed paraffin-embedded (FFPE) breast cancer cells and tissue blocks. This study represents the first ever demonstration of an aptamer against progesterone receptor and its diagnostic capacity.

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Monitoring health-related biomarkers using fast and facile detection techniques provides key physicochemical information for disease diagnosis or reflects body health status. Among them, electrochemical detection of various bio-macromolecules, e.g., the C-reactive protein (CRP), is of great interest in offering potential diagnosis for acute inflammation caused by infections, heart diseases, etc. Herein, a novel electrochemical aptamer biosensor was constructed from Ti3C2Tx MXene and in-situ reduced Au NPs for thiolated-RNA aptamer immobilization and CRP protein detection using Fc(COOH) as the signal probe. The sensory performances for CRP detection were optimized based on working conditions, including the incubation times and the pH. The large surface area offered by Ti3C2Tx MXene and high electrical conductivity originating from Au NPs endowed the as-fabricated aptamer biosensor with a decent sensitivity for CRP in a wide linear range of 0.05-80.0 ng/mL, good selectivity over interfering substances, and a low detection limit of 0.026 ng/mL. Such aptamer biosensors also detected CRP in serum samples using the spike & recovery method with reasonable recovery rates. The results demonstrated the potential of the as-fabricated electrochemical aptamer biosensor for fast and facile CRP detection in practical applications.

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The outbreak of SARS-CoV-2 poses a serious threat to human life and health. A rapid nucleic acid tests can effectively curb the spread of the disease. With the advantages of fluorescent RNA aptamers, low background and high sensitivity. A variety of fluorescent RNA aptamer sensors have been developed for the detection of nucleic acid. Here, we report a hypersensitive detection platform in which SARS-CoV-2 initiates RTF-EXPAR to amplify trigger fragments. This activation leads to the reassembled of the SRB2 fluorescent RNA aptamer, restoring its secondary structure for SR-DN binding and turn-on fluorescence. The platform completes the assay in 30 min and all reactions occur in one tube. The detection limit is as low as 116 aM. Significantly, the platform's quantitative analyses were almost identical to qPCR results in simulated tests of positive samples. In conclusion, the platform is sensitive, accurate and provides a new protocol for point-of-care testing of viruses.

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RNA synthetic biology tools have primarily been applied in E. coli; however, many other bacteria are of industrial and clinical significance. Thus, the multicolor fluorogenic aptamer Pepper was evaluated in both Gram-positive and Gram-negative bacteria. Suitable HBC-Pepper dye pairs were identified that give blue, green, or red fluorescence signals in the E. coli, Bacillus subtilis, and Salmonella enterica serovar Typhimurium (S. Typhimurium). Furthermore, we found that different RNA scaffolds have a drastic effect on in vivo fluorescence, which did not correlate with the in vitro folding efficiency. One such scaffold termed DF30-tRNA displays 199-fold greater fluorescence than the Pepper aptamer alone and permits simultaneous dual color imaging in live cells.

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RNAs play important roles in regulating biological growth and development. Advancements in RNA-imaging techniques are expanding our understanding of their function. Several common RNA-labeling methods in plants have pros and cons. Simultaneously, plants' spontaneously fluorescent substances interfere with the effectiveness of RNA bioimaging. New technologies need to be introduced into plant RNA luminescence. Aggregation-induced emission luminogens (AIEgens), due to their luminescent properties, tunable molecular size, high fluorescence intensity, good photostability, and low cell toxicity, have been widely applied in the animal and medical fields. The application of this technology in plants is still at an early stage. The development of AIEgens provides more options for RNA labeling. Click chemistry provides ideas for modifying AIEgens into RNA molecules. The CRISPR/Cas13a-mediated targeting system provides a guarantee of precise RNA modification. The liquid-liquid phase separation in plant cells creates conditions for the enrichment and luminescence of AIEgens. The only thing that needs to be looked for is a specific enzyme that uses AIEgens as a substrate and modifies AIEgens onto target RNA via a click chemical reaction. With the development and progress of artificial intelligence and synthetic biology, it may soon be possible to artificially synthesize or discover such an enzyme.

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S-Adenosyl methionine (SAM) is a critical metabolite involved in numerous cellular processes, including DNA methylation and gene expression regulation. Understanding the spatiotemporal dynamics of SAM within living cells is essential for deciphering its roles in maintaining cell homeostasis and in disease development. Here, we describe a protocol based on a recently reported SAM sensor exploiting a fluorogenic RNA and an RNA three-way junction for visualizing SAM dynamics in cultured mammalian cells.

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BACKGROUND: Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the most abundant soluble protein in nature. Extensive studies have been conducted for improving its activity in photosynthesis through approaches like protein engineering. Concurrently, multiple biochemical and radiolabeling assays have been developed for determining its activity. Although these existing assays yield reliable results, they require addition of multiple external components, rendering them less convenient and expensive. Therefore, in this study, we have developed two relatively cheaper, convenient, and easily reproducible assays for quantitative and qualitative estimation of RuBisCO activity. RESULTS: We simplified a contemporary NADH based spectrophotometric RuBisCO assay by using cyanobacterial cell lysate as the source for Calvin cycle enzymes. We analyzed the influence of inorganic carbon substrates, CO2 and NaHCO3, and varying protein concentrations on RuBisCO activity. Ribulose-1,5-bisphosphate (RuBP) consumption rates for the cultures grown under 5% CO2 were 5-7 times higher than the ones grown with 20 mM NaHCO3, at different protein concentrations. The difference could be due to the impaired activity of carbonic anhydrase in the cell lysate, which is required for the conversion of HCO3- to CO2. The highest RuBisCO activity of 2.13 nmol of NAD+/ µg of Chl-a/ min was observed with 50 µg of protein and 5% CO2. Additionally, we developed a novel RNA-sensor based fluorescence assay that is based on the principle of tracking the kinetics of ATP hydrolysis to ADP during the conversion of 3-phosphoglycerate (3-PG) to 1,3-bisphosphoglycerate (1,3-BPG) in the Calvin cycle. Under in vitro conditions, the fluorometric assay exhibited  ~ 3.4-fold slower reaction rate (0.37 min-1) than the biochemical assay when using 5% CO2. We also confirmed the in vivo application of this assay, where increase in the fluorescence was observed with the recombinant strain of Synechocystis sp. PCC 6803 (SSL142) expressing the ADP-specific RNA sensor, compared to the WT. In addition, SSL142 exhibited three-fold higher fluorescence when supplemented with 20 mM NaHCO3 as compared to the cells that were grown without NaHCO3 supplementation. CONCLUSIONS: Overall, we have developed a simplified biochemical assay for monitoring RuBisCO activity and demonstrated that it can provide reliable results as compared to the prior literature. Furthermore, the biochemical assay using 5% CO2 (100% relative activity) provided faster RuBP consumption rate compared to the biochemical assay utilizing 20 mM NaHCO3 (30.70% relative activity) and the in vitro fluorometric assay using 5% CO2 (29.64% relative activity). Therefore, the absorbance-based biochemical assay using 5% CO2 or higher would be suitable for in vitro quantification of the RuBisCO activity. On the other hand, the RNA-sensor based in vivo fluorometric assay can be applied for qualitative analysis and be used for high-throughput screening of RuBisCO variants. As RuBisCO is an enzyme shared amongst all the photoautotrophs, the assays developed in this study can easily be extended for analyzing the RuBisCO activities even in microalgae and higher plants.

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Throughout their entire life cycle, RNAs are associated with RNA-binding proteins (RBPs), forming ribonucleoprotein (RNP) complexes with highly dynamic compositions and very diverse functions in RNA metabolism, including splicing, translational regulation, ribosome assembly. Many RNPs remain poorly characterized due to the challenges inherent in their purification and subsequent biochemical characterization. Therefore, developing methods to isolate specific RNA-protein complexes is an important initial step toward understanding their function. Many elegant methodologies have been developed to isolate RNPs. This chapter describes different approaches and methods devised for RNA-specific purification of a target RNP. We focused on general methods for selecting RNPs that target a given RNA under conditions favourable for the copurification of associated factors including RNAs and protein components of the RNP.

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TDP-43 is an aggregation-prone protein which accumulates in the hallmark pathological inclusions of amyotrophic lateral sclerosis (ALS). However, the analysis of deeply phenotyped human post-mortem samples has shown that TDP-43 aggregation, revealed by standard antibody methods, correlates poorly with symptom manifestation. Recent identification of cryptic-splicing events, such as the detection of Stathmin-2 (STMN-2) cryptic exons, are providing evidence implicating TDP-43 loss-of-function as a potential driving pathomechanism but the temporal nature of TDP-43 loss and its relation to the disease process and clinical phenotype is not known. To address these outstanding questions, we used a novel RNA aptamer, TDP-43APT, to detect TDP-43 pathology and used single molecule in situ hybridization to sensitively reveal TDP-43 loss-of-function and applied these in a deeply phenotyped human post-mortem tissue cohort. We demonstrate that TDP-43APT identifies pathological TDP-43, detecting aggregation events that cannot be detected by classical antibody stains. We show that nuclear TDP-43 pathology is an early event, occurring prior to cytoplasmic accumulation and is associated with loss-of-function measured by coincident STMN-2 cryptic splicing pathology. Crucially, we show that these pathological features of TDP-43 loss-of-function precede the clinical inflection point and are not required for region specific clinical manifestation. Furthermore, we demonstrate that gain-of-function in the form of extensive cytoplasmic accumulation, but not loss-of-function, is the primary molecular correlate of clinical manifestation. Taken together, our findings demonstrate implications for early diagnostics as the presence of STMN-2 cryptic exons and early TDP-43 aggregation events could be detected prior to symptom onset, holding promise for early intervention in ALS.

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In this study, we present a one-pot, one-step, label-free miRNA detection method through a structural transition of a specially designed dumbbell-shape probe, initiating a rolling circle transition (RCT). In principle, target miRNA binds to right loop of the dumbbell probe (DP), which allows structural change of the DP to circular form, exposing a sequence complementary to the T7 promoter (T7p) previously hidden within the stem. This exposure allows T7 RNA polymerase to initiate RCT, producing a repetitive Mango aptamer sequence. TO1-biotin, fluorescent dye, binds to the aptamer, inducing a detectable enhancement of fluorescence intensity. Without miR-141, the DP stays closed, RCT is prevented, and the fluorescence intensity remains low. By employing this novel strategy, target miRNA was successfully identified with a detection of 73 pM and a dynamic linear range of 0-10 nM. Additionally, the method developed enables one-pot, one-step, and label-free detection of miRNA, demonstrating potential for point-of-care testing (POCT) applications. Furthermore, the practical application of the designed technique was demonstrated by reliably detecting the target miRNA in the human serum sample. We also believe that the conceived approach could be widely used to detect not only miRNAs but also diverse biomolecules by simply replacing the detection probe.

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DNA methyltransferase is significant in cellular activities and gene expression, and its aberrant expression is closely linked to various cancers during initiation and progression. Currently, there is a great demand for reliable and label-free techniques for DNA methyltransferase evaluation in tumor diagnosis and cancer therapy. Herein, a low-background fluorescent RNA aptamer-based sensing approach for label-free quantification of cytosine-guanine (CpG) dinucleotides methyltransferase (M.SssI) is reported. The fluorogenic light-up RNA aptamers-based strategy exhibits high selectivity via restriction endonuclease, padlock-based recognition, and RNA transcription. By combining rolling circle amplification (RCA), and RNA transcription with fluorescence response of RNA aptamers of Spinach-dye compound, the proposed platform exhibited efficiently ultrahigh sensitivity toward M.SssI. Eventually, the detection can be achieved in a linear range of 0.02-100 U mL-1 with a detection limit of 1.6 × 10-3 U mL-1. Owing to these superior features, the method is further applied in serum samples spiked M.SssI, which delivers a recovery ranging from 92.0 to 107.0% and a relative standard deviation <7.0%, providing a promising and practical tool for determining M.SssI in complex biological matrices.

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The abnormal expression levels of miRNAs have been proven to be highly related to the generation of various diseases and are also closely associated with the stages and types of disease development. The novel RNA aptamers-based homogenous fluorescent methods were simple, with low background signal and high signal-to-noise ratio, but lacked effective signal amplification technology to achieve sensitive detection of trace miRNA markers. There is an urgent need for combining effective nucleic acid amplification technology with RNA aptamer to achieve highly sensitive and accurate detection of miRNA. For this purpose, a new DNA multi-arm nanostructure-based dual rolling circle transcription machinery for the generation of lighting-up MG RNA aptamers is constructed for label-free and highly sensitive sensing of miRNA-21. In this system, the target miRNA-21 induces a structural transformation of the DNA multi-arm nanostructure probe to recycle miRNA-21 and trigger two independent rolling circle transcription reactions to generate two long RNAs, which can partially hybridize with each other to generate large amounts of complete MG RNA aptamers. These RNA aptamers can associate with organic MG dye to produce significantly enhanced fluorescence signals to accomplish ultrasensitive miRNA-21 detection down to 0.9 fM. In addition, this method exhibits high selectivity to distinguish miRNA-21 even with single nucleotide mismatch, and also has potential application capability to monitor different expression levels of miRNA-21 from different cancer cells. The effective collaboration between MG RNA aptamer and rolling circle transcription reaction makes this fluorescent method show the significant advantages of low background signal, high signal-to-noise ratio and high detection sensitivity. It has great potential to be a promising means to achieve label-free and highly sensitive monitoring of other trace biological markers via a simple change of target sequence.

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