Beacon Design Research Articles

NanoMIP beacons with a co-operative binding mechanism for the all-in-one detection of methamphetamine aptamer complexes

Methamphetamine is a highly addictive stimulant with significant public health implications, necessitating the development of rapid, sensitive, and reliable detection methods. Traditional analytical techniques, though accurate, often involve complex sample preparation, expensive equipment, and lengthy analysis times. This study presents the design, synthesis, and application of nanoMIP beacons with a unique co-operative binding mechanism for the detection of methamphetamine. NanoMIP beacons selective for methamphetamine/aptamer complexes were synthesized using a solid-phase synthesis method that involved the bio-conjugation of an aptamer on the stationary phase and the incubation of methamphetamine to form a methamphetamine-aptamer complex. The resultant nanoMIP beacons were eluted off the affinity column and characterised using transmission electron microscopy. The co-operative binding mechanism, which relies on the structure-switching capability of the aptamer upon analyte binding was demonstrated through comparison of the fluorescence quenching signal of a scrambled sequence and nanoNIP controls. The fluorescence quenching assay was established using a fixed optimal concentration of aptamer and varying amounts of methamphetamine. The nanoMIP beacons showed enhanced the sensitivity (LOD = 23 ± 3.5 nM) and excellent selectivity, with a 40-fold increase in quenching for methamphetamine compared to other illicit drugs. The nanoMIP beacons demonstrated acceptable sample recoveries in both urine diluent and 50% human serum. This work provides a new strategy for the development of hybrid nanoMIP/aptamer-based sensors and provides a robust analytical tool for combating methamphetamine abuse.

Real-time quantitative reverse transcription PCR assay for the detection of Nuomin virus – An emerging tick-borne virus

Nuomin virus (NOMV), an emerging tick-borne virus (TBVs) identified in 2020, has been associated with fever, headache, and potential liver dysfunction in infected individuals. This study presents a novel TaqMan real-time quantitative PCR method designed for the rapid, sensitive, and specific detection of NOMV, facilitating early diagnosis. Utilizing Beacon Designer software 8.0, we optimized the PCR assay including the development of primers and probes to precisely target the conserved region of the NOMV genome, followed by optimization of primer and probe concentrations and annealing temperature. The resulting assay demonstrated robust performance, with standard curve represented by the equation y=−3.29x+39.42, a high correlation coefficient (R2 = 0.995) and an efficiency 99.53 %. Importantly, the method exhibited exceptional specificity, which did not yield cross-reacting signals from other TBVs, including Songling virus (SGLV), Beiji virus (BJNV), tick-borne encephalitis virus (TBEV), Yezo virus (YEZV), Alongshan virus (ALSV), and severe fever with thrombocytopenia syndrome bunyavirus (SFTSV). The assay’s detection limit was remarkably low, reaching 10 copies/μL, representing a 100-fold increase compared to semi-nested RT-PCR. Additionally, it demonstrated excellent repeatability, with coefficients of variation for intra- and inter-group tests consistently below 3 %. Clinical evaluations confirmed the assay’s superior performance, highlighting its high specificity, sensitivity, and reproducibility for NOMV detection. In conclusion, the method developed in this study provides a valuable tool to support timely management of NOMV infections, with significant implications for clinical practice.

A molecular beacon design for a colorimetric loop-mediated isothermal amplification assay.

In this study, a molecular beacon (MB) was designed for colorimetric loop-mediated isothermal amplification (cLAMP). The length of complementary bases on the MB, guanine and cytosine content (GC content), and hybridization sites of complementary bases were investigated as key factors affecting the design of the MB. We designed MBs consisting of 10, 15, and 20 complementary bases located at both ends of the HRPzyme. In the case of the long dumbbell DNA structure amplified from the hlyA gene of Listeria monocytogenes, possessing a flat region (F1c-B1) of 61base pairs (bp), an MB was designed to intercalate into the flat region between the F1c and B1 regions of the LAMP amplicons. In the case of the short dumbbell DNA structure amplified from the bcfD gene of Salmonella species possessing a flat region (F1c-B1) length of 6bp, another MB was designed to intercalate into the LoopF or LoopB regions of the LAMP amplicons. The results revealed that the hybridization site of the MB on the LAMP amplicons was not crucial in designing the MB, but the GC content was an important factor. The highest hybridization efficiencies for LAMP amplicons were obtained from hlyA gene-specific and bcfD gene-specific MBs containing 20- and 15-base complementary sequences, respectively, which exhibited the highest GC content. Therefore, designing MBs with a high GC content is an effective solution to overcome the low hybridization efficiency of cLAMP assays. The results obtained can be used as primary data for designing MBs to improve cLAMP accessibility.

High-Fidelity Sensitive Tracing Circulating Tumor Cell Telomerase Activity.

Dynamic tracing of intracellular telomerase activity plays a crucial role in cancer cell recognition and correspondingly in earlier cancer diagnosis and personalized precision therapy. However, due to the complexity of the required reaction system and insufficient loading of reaction components into cells, achieving a high-fidelity determination of telomerase activity is still a challenge. Herein, an Aptamer-Liposome mediated Telomerase activated poly-Molecular beacon Arborescent Nanoassembly(ALTMAN) approach was described for direct high-fidelity visualization of telomerase activity. Briefly, intracellular telomerase activates molecular beacons, causing their hairpin structures to unfold and produce fluorescent signals. Furthermore, multiple molecular beacons can self-assemble, forming arborescent nanostructures and leading to exponential amplification of fluorescent signals. Integrating the enzyme-free isothermal signal amplification successfully increased the sensitivity and reduced interference by leveraging the skillful design of the molecular beacon and the extension of the telomerase-activated TTAGGG repeat sequence. The proposed approach enabled ultrasensitive visualization of activated telomerase exclusively with a prominent detection limit of 2 cells·μL-1 and realized real-time imaging of telomerase activity in living cancer cells including blood samples from breast cancer patients and urine samples from bladder cancer patients. This approach opens an avenue for establishing a telomerase activity determination and in situ monitoring technique that can facilitate both telomerase fundamental biological studies and cancer diagnostics.

Turn-on RNA Mango Beacons for trans-acting fluorogenic nucleic acid detection.

The Mango I and II RNA aptamers have been widely used in vivo and in vitro as genetically encodable fluorogenic markers that undergo large increases in fluorescence upon binding to their ligand, TO1-Biotin. However, while studying nucleic acid sequences, it is often desirable to have trans-acting probes that induce fluorescence upon binding to a target sequence. Here, we rationally design three types of light-up RNA Mango Beacons based on a minimized Mango core that induces fluorescence upon binding to a target RNA strand. Our first design is bimolecular in nature and uses a DNA inhibition strand to prevent folding of the Mango aptamer core until binding to a target RNA. Our second design is unimolecular in nature, and features hybridization arms flanking the core that inhibit G-quadruplex folding until refolding is triggered by binding to a target RNA strand. Our third design builds upon this structure, and incorporates a self-inhibiting domain into one of the flanking arms that deliberately binds to, and precludes folding of, the aptamer core until a target is bound. This design separates G-quadruplex folding inhibition and RNA target hybridization into separate modules, enabling a more universal unimolecular beacon design. All three Mango Beacons feature high contrasts and low costs when compared to conventional molecular beacons, with excellent potential for in vitro and in vivo applications.

Rethinking infrastructure design: evaluating pedestrians and VRUs’ psychophysiological and behavioral responses to different roadway designs

The integration of human-centric approaches has gained more attention recently due to more automated systems being introduced into our built environments (buildings, roads, vehicles, etc.), which requires a correct understanding of how humans perceive such systems and respond to them. This paper introduces an Immersive Virtual Environment-based method to evaluate the infrastructure design with psycho-physiological and behavioral responses from the vulnerable road users, especially for pedestrians. A case study of pedestrian mid-block crossings with three crossing infrastructure designs (painted crosswalk, crosswalk with flashing beacons, and a smartphone app for connected vehicles) are tested. Results from 51 participants indicate there are differences between the subjective and objective measurement. A higher subjective safety rating is reported for the flashing beacon design, while the psychophysiological and behavioral data indicate that the flashing beacon and smartphone app are similar in terms of crossing behaviors, eye tracking measurements, and heart rate. In addition, the smartphone app scenario appears to have a lower stress level as indicated by eye tracking data, although many participants do not have prior experience with it. Suggestions are made for the implementation of new technologies, which can increase public acceptance of new technologies and pedestrian safety in the future.

Nucleobase intrinsic quenchers: A fluorescence off switch

Molecular beacons are important bioanalytical probes most often constructed from a single-stranded oligonucleotide which has been labeled at opposite termini with a fluorophore and a quencher. When the fluorophore and quencher are in close proximity, ideally no fluorescence is observed due to Fluorescence Resonance Energy Transfer (FRET). 4-Dimethylaminoazobenzene-4′-carboxylic acid (DABCYL) is a commonly used quencher in molecular beacons; however, DABCYL is unable to form a base-pair and is conventionally installed as an overhanging residue. In this arrangement, the DABCYL moiety has substantial mobility which affects the accuracy of FRET-related distance/orientation measurements and also limits the types of other conjugates that can be prepared. To address these limitations, we have synthesized peptide nucleic acid (PNA) analogues possessing DMPAU (5-[(4-dimethylaminophenyl)diazenyl]uracil) and NPhpC (6-(4-nitrophenyl)pyrrolocytosine) nucleobases which act as dark fluorophores, i.e. quenchers, while retaining the ability to base pair by canonical hydrogen bonding. This will allow more flexible design and construction of molecular beacons based on the insertion of nucleobase fluorophores and quenchers into the stem sequence. We have quantified the quenching ability of DMPAU and NPhpC PNA analogues by determining the Stern-Volmer constants against the intrinsically fluorescent nucleobase, phenylpyrrolocytosine, and other blue emitting fluorophores (pyrene, acridone). The hydrogen bonding ability of DMPAU with adenine, as determined by 1H NMR titration, is relatively unperturbed compared to uracil and thymine.

Tracking Small Wildlife With Minimal-Complexity Radio Frequency Transmitters: Near-Optimal Detection

In a range of IoT applications, edge nodes require minimal size, mass, and power/energy use. When node complexity is severely constrained, the burden of detection falls to the receiving system. In this paper we present a method of detection for RF signals motivated by wildlife radio telemetry, where miniature beacons emit VHF pulses that are used to detect and localize animals in the field. We address the challenges of limited transmit power and path losses due to terrain that lead to low received SNR. In addition, the pulse train structure requires joint frequency acquisition, time synchronization, and detection; our approach addresses the key challenge of large uncertainty in signal parameters. Our algorithm explicitly integrates frequency acquisition and time synchronization, using nearly phase-coherent matched filtering followed by non-coherent pulse combining and a multiple hypotheses test to provide near-optimal detection performance in low SNR scenarios. We provide quantitative results showing performance as a function of SNR and signal uncertainty. When compared to the single pulse detection case as used in existing systems, our multipulse combining approach pulses improves detection performance by 4 dB, even under the conditions of temporal and frequency/phase uncertainty resulting from beacon design and manufacturing. We also describe an efficient real-time signal processing implementation of the algorithm, including signal tracking that supports localization after initial signal detection.

Ionospheric measurement using a linearly polarized tri-band small satellite beacon

Radio waves propagating the ionosphere are subject to a number of unpredictable and corrupting effects, the most significant of which are phase path reduction, Faraday rotation, and scintillation. A method of measuring these effects is necessary in order to study and understand the solar-terrestrial connections, as well as to anticipate their impact on radio systems. However, limitations in current measurement solutions, most notably the lack of direct ionospheric Faraday rotation measurement capability, restricts the ability to characterize the ionosphere to a sufficient degree for applications such as the detection of coronal mass ejections. The rise in popularity of small satellites presents a new cost-effective opportunity to study and observe the ionosphere in detail, however the challenge of realizing a beacon within the constraints of a small satellite system must be overcome. In this paper, we introduce a measurement technique that builds on past multi-frequency beacons, incorporating the well established differential phase technique, as well as introducing a new differential polarization technique. We achieve this by employing linearly polarized beacon signals, which enables us to separate phase path reduction and Faraday rotation into a phase and polarization error respectively. The introduction of linear polarization overcomes a key limitations in current ionospheric measurement techniques: eliminating errors associated with indirect Faraday rotation measurement. The additional polarization data also enables absolute unambiguous measurement of both parameters with fewer and lower frequencies than conventional solutions, as well as without the need for precise satellite clock, phase biases and positioning data. These characteristics correspond with a reduction in the size, power and complexity of the satellite payload, and as a result, our technique better suited to small satellite applications. To validate the capability of measuring the ionosphere within the constraints of a small satellite package, we establish the influence of system noise, weak scattering, clock errors and antenna characteristics on the measurement technique. We then use this to inform a starting point for a CubeSat beacon and receiver design solution which achieves unambiguous and high-resolution measurement of ionospheric parameters with practicable system requirements and constraints. This starting point demonstrates that a resolution of 12mTECU and 0.03rad/m2 for the electron density and Faraday rotation estimates respectively, as well as useful scintillation measurements dominated by scintillation noise may be achieved utilizing a tri-band 3U CubeSat beacon with frequencies 144.2MHz, 432.6MHz, and 1297.8MHz, a transmitter power of no more than 4watts, and antenna requirements consistent with a dipole type antenna.

Dual electroactive AgM (M=Ru, Pt) NPs for double electroanalysis of HER2 and EpCAM

Parallel multiple detections of tumor markers is of significant for the efficient and accurate early monitoring of related cancers. Electrochemical aptasensors possess excellent selectivity and sensitive quantitative detection while maintaining multiple detection ability. The study of high and different electrochemical beacons is urgent to achieve multiple-level parallel detection. In this work, electroactive AgRu NPs and AgPt NPs were explored to exhibit electrooxidation peaks at 0.8 V and − 0.7 V, respectively. Plasmonic AgRu NPs and AgPt NPs harvested light energy and exhibited amplified electrooxidation activity under local surface plasmon resonance (LSPR) excitation. Plasmon-enhanced electrooxidation principle was proposed for AgM (M=Ru, Pt) NPs owing to the distinct imaginary part of dielectric constants between Ag and platinum group metals (PGM). The strong and non-interference electrooxidation peaks at 0.8 V and − 0.7 V enabled AgRu NPs and AgPt NPs to be different electroactive beacons for the sensitive and reliable quantitative dual detection of human epidermal growth factor receptor-2 (HER2) and epithelial cell adhesion molecule (EpCAM). The LODs were 0.3 pg mL−1 (7.5 amol mL−1) for EpCAM and 0.6 pg mL−1 (3.2 amol mL−1) for HER2. The proposed plasmon-promoted electrooxidation principle provides a novel route for the design of multiple electroactive plasmonic metal-PGM composite electrochemical beacons for simultaneous electroanalysis applications.

ASSESSMENT OF 3D POSITIONAL ACCURACY OF GEODETIC OBSERVATIONS FROM SINGLE CORS

Geodetic observations for both vertical and horizontal control networks cannot be compromised for any reason in accuracy and precision in the field of geomatics. Due to the error-prone nature of survey measurements, standards are established to allow for comparing the obtained results with a set of guidelines, regulations, or pre-determined specifications. The University of Benin’s Ugbowo Campus in Nigeria does not have enough control points, which informs this study. The densification of more reliable control points using the most recent technology is necessary. Before the observations, control network design, excavation, casting, and monumentation of first-order compliance beacons had been completed. Eight GNSS receivers were connected to the CORS_Geosystems multi-link access point and simultaneously deployed for observations. The stages involve the adjustment of observed data, the presentation of adjusted results, and the determination of horizontal and vertical accuracies. The result of horizontal accuracy showed that the RAPH_GNSS_08 station had the highest horizontal accuracy standard ratio of 1:432,193, while the Raph_GNSS_04 station had the lowest, 1:133,271. The highest vertical accuracy standard was 4.0mm, achieved between Cors_Geo and RAPH_GNSS_09, while the lowest, which was 3.1mm, was observed between Cors_Geo and RAPH_GNSS_08. High-precision engineering projects in the research area will benefit from the established first-order controls in terms of execution, monitoring, and maintenance. The Surveyors Council of Nigeria (SURCON) has recommended GNSS as one of the methods for achieving geodetic control densification in Nigeria.

In silico SYBR® green-based qPCR primer design for virulent protein C gene of Edwardsiella tarda

Edwardsiella tarda infection in fish results in economic losses in aquaculture industries worldwide . Besides, this pathogen is also a zoonotic agent causing gastrointestinal disease in humans. Detection of virulence factors expressed by E. tarda is a key for molecular diagnosis. This study aimed to design SYBR ® Green-based quantitative PCR (qPCR) primer for E. tarda virulent protein C (EvpC) gene. A sequence of EvpC with accession number AY424360.4:6500-6991 obtained from GenBank NCBI was selected as the basis for qPCR primer design. qPCR primers were designed using Primer3 online software. Further analysis related to the secondary structure of each primer was carried out using Beacon Designer Free Edition. Five pairs of qPCR primers were successfully designed with Primer3. Based on the results of Primer3 and Beacon Designer analysis, F primer pair 1 (5'-GAATCCACCGACGATAAGCACAAA-3') and R primer pair 1 (5'-GACACGCAGCACCGACATCA-3') were the most favorable primer set since this primer met the criteria in terms of length, melting temperature, GC content, and self complementarity. In addition, this primer set had the highest (closest to zero) - ΔG value of cross dimer, self dimer, and hairpin. It can be concluded that SYBR ® Green-based qPCR primer set for EvpC has been successfully designed. However, laboratory optimization experiments are essential for this designed primer set.

A Design of COSPAS-SARSAT Beacon with BDS Return Link Service

This article describes a proto-type beacon, which supports search and rescue (SAR) return link service (RLS) provided by BeiDou Satellite Navigation System (BDS). According to the design of signal-in-space format of BDS RLS, with related technologies from COSPAS-SARSAT, comparing with Galileo RLS system, this article describes a design of BDS RLS beacon proto-type, and how it receives and processes return link message (RLM). Then three interfaces are analysed, which are the GNSS signal interface from the BDS satellite to the GNSS receiver, the GNSS receiver sentence interface from the GNSS receiver to the beacon processing unit and the human-machine interaction interface for the beacon to the user. Finally, its test result and cost-performance trade-off are discussed.

Detection of SARS-CoV-2 RNA using RT-LAMP and molecular beacons

BackgroundRapid spread of SARS-CoV-2 has led to a global pandemic, resulting in the need for rapid assays to allow diagnosis and prevention of transmission. Reverse transcription-polymerase chain reaction (RT-PCR) provides a gold standard assay for SARS-CoV-2 RNA, but instrument costs are high and supply chains are potentially fragile, motivating interest in additional assay methods. Reverse transcription and loop-mediated isothermal amplification (RT-LAMP) provides an alternative that uses orthogonal and often less expensive reagents without the need for thermocyclers. The presence of SARS-CoV-2 RNA is typically detected using dyes to report bulk amplification of DNA; however, a common artifact is nonspecific DNA amplification, which complicates detection.ResultsHere we describe the design and testing of molecular beacons, which allow sequence-specific detection of SARS-CoV-2 genomes with improved discrimination in simple reaction mixtures. To optimize beacons for RT-LAMP, multiple locked nucleic acid monomers were incorporated to elevate melting temperatures. We also show how beacons with different fluorescent labels can allow convenient multiplex detection of several amplicons in “single pot” reactions, including incorporation of a human RNA LAMP-BEAC assay to confirm sample integrity. Comparison of LAMP-BEAC and RT-qPCR on clinical saliva samples showed good concordance between assays. To facilitate implementation, we developed custom polymerases for LAMP-BEAC and inexpensive purification procedures, which also facilitates increasing sensitivity by increasing reaction volumes.ConclusionsLAMP-BEAC thus provides an affordable and simple SARS-CoV-2 RNA assay suitable for population screening; implementation of the assay has allowed robust screening of thousands of saliva samples per week.

Label-Free Fluorescence Molecular Beacon Probes Based on G-Triplex DNA and Thioflavin T for Protein Detection.

Protein detection plays an important role in biological and biomedical sciences. The immunoassay based on fluorescence labeling has good specificity but a high labeling cost. Herein, on the basis of G-triplex molecular beacon (G3MB) and thioflavin T (ThT), we developed a simple and label-free biosensor for protein detection. The biotin and streptavidin were used as model enzymes. In the presence of target streptavidin (SA), the streptavidin hybridized with G3MB-b (biotin-linked-G-triplex molecular beacon) perfectly and formed larger steric hindrance, which hindered the hydrolysis of probes by exonuclease III (Exo III). In the absence of target streptavidin, the exonuclease III successively cleaved the stem of G3MB-b and released the G-rich sequences which self-assembled into a G-triplex and subsequently activated the fluorescence signal of thioflavin T. Compared with the traditional G-quadruplex molecular beacon (G4MB), the G3MB only needed a lower dosage of exonuclease III and a shorter reaction time to reach the optimal detection performance, because the concise sequence of G-triplex was good for the molecular beacon design. Moreover, fluorescence experiment results exhibited that the G3MB-b had good sensitivity and specificity for streptavidin detection. The developed label-free biosensor provides a valuable and general platform for protein detection.

A Genetic Code Expansion-Derived Molecular Beacon for the Detection of Intracellular Amyloid-β Peptide Generation.

Polypeptides generated from proteolytic processing of protein precursors, or proteolytic proteoforms, play an important role in diverse biological functions and diseases. However, their often‐small size and intricate post‐translational biogenesis preclude the use of simple genetic tagging in their cellular studies. Herein, we develop a labeling strategy for this class of proteoforms, based on residue‐specific genetic code expansion labeling with a molecular beacon design. We demonstrate the utility of such a design by creating a molecular beacon reporter to detect amyloid‐β peptides, known to be involved in the pathogenesis of Alzheimer's disease, as they are produced from amyloid precursor protein (APP) along the endocytic pathway of living cells.

A Genetic Code Expansion-Derived Molecular Beacon for the Detection of Intracellular Amyloid-β Peptide Generation.

Polypeptides generated from proteolytic processing of protein precursors, or proteolytic proteoforms, play an important role in diverse biological functions and diseases. However, their often-small size and intricate post-translational biogenesis preclude the use of simple genetic tagging in their cellular studies. Herein, we develop a labeling strategy for this class of proteoforms, based on residue-specific genetic code expansion labeling with a molecular beacon design. We demonstrate the utility of such a design by creating a molecular beacon reporter to detect amyloid-β peptides, known to be involved in the pathogenesis of Alzheimer's disease, as they are produced from amyloid precursor protein (APP) along the endocytic pathway of living cells.

Primer parameters defining efficiency and coefficient of determination in quantitative polymerase chain reaction

We performed a correlation analysis between primer parameters and qPCR efficiency/coefficient of determination in two independent samples from in vitro functional experiments.Primer parameters do not define qPCR efficiency and coefficient of determination significantly if primers are designed according to the optimised PRIMER-BLAST settings.Aim. To find the correlation between the primer parameters, efficiency, and coefficient of determination (R2 ) in quantitative polymerase chain reaction (qPCR) conditions.Methods. Upon RNA isolation from primary human coronary artery endothelial cells, we performed reverse transcription-qPCR (RT-qPCR) utilising SYBR Green chemistry to measure the expression of the following genes: IL1B, IL6, CXCL8, IL12A, IL23A, PECAM1, VWF, KDR, FAPA, ACTA2, SMTN, VIM, COL4A1, MMP2, SNAI2, TWIST1, ZEB1, SCARF1, CD36, LDLR, VLDLR, VCAM1, ICAM1, SELE, SELP, CDH5, IL1R1, IL1R2, TNFRSF1A, TNFRSF1B, NOS3, PXDN. Primers were designed employing Primer-BLAST software using optimised settings. For the correlation analysis, Spearman's rank correlation coefficient was applied (GraphPad Prism).Results. Coefficient of determination correlated with the primer pair rating by Beacon Designer, amplicon melting temperature, and GC content in the reverse primer. Reaction efficiency did not correlate with the Beacon Designer rating, yet being associated with length and GC content of the reverse primer. Abovementioned correlation coefficients ranged from 0.4 to 0.5 or from -0.4 to -0.5 indicative of moderate positive or negative correlation. Other parameters did not affect reaction efficiency and coefficient of determination. Conclusion Primer parameters do not define qPCR efficiency and coefficient of determination significantly if primers are designed according to the optimised PRIMER-BLAST settings.

A Spherical Nucleic Acid Probe Based on the Au-Se Bond.

Spherical nucleic acid probes (SNAPs) are 3D nucleic acid nanostructures with multiple superiorities over bare nucleic acid chains. Au-based SNAPs that employ gold nanoparticles (AuNPs) as cores and densely modified nucleic acid chains (commonly via Au-S bonds) as shells have been extensively investigated for the diagnosis and therapy of diverse diseases. However, abundant biothiols in living cells can severely displace nucleic acid chains from AuNPs and restrict their theranostic performance. Herein we report the design and preparation of a selenol terminal-functionalized molecular beacon (MB-SeH), which was further employed to prepare a Au-Se bond-based SNAP (SNAP-Se) for bioimaging. A series of experiments proved the successful preparation of MB-SeH and SNAP-Se, and the obtained nanoprobe could avoid biothiol interference and eliminate the false positive signals during biomarker imaging in living cells. This work will open a new avenue for the design and application of SNAPs.

Free Solution Assay Signal Modulation in Variable-Stem-Length Hairpin Aptamers.

Interferometric measurements of free solution assays (FSAs) quantify changes in molecular conformation and hydration upon binding. Here, we demonstrate that aptamer probes designed to undergo varying levels of conformational change upon binding produce corresponding variations in FSA signals. A series of hairpin aptamers were synthesized for the small molecule (tenofovir) with identical loop regions that contain the binding pocket, with between 2 and 10 self-associating base pairings in the stem region. Aptamers selected for tenofovir showed a decrease in the FSA signal and binding affinity (increase in KD) with increasing stem length. Thermodynamic calculations of the Gibbs free energy (ΔG) reported a decrease in ΔG with respect to a corresponding increase in the aptamer stem length. Collectively, these observations provide an expanded understanding of FSA and demonstrate the potential for the rational design of label-free aptamer beacons using FSA as readout.