fSHAPE, fSHAPE-eCLIP, and SHAPE-eCLIP probe transcript regions that interact with specific proteins.

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) structure probing techniques characterize the secondary structure of RNA molecules, which influence their functions and interactions. A variation of SHAPE, footprinting SHAPE (fSHAPE), probes RNA in the presence and absence of protein to identify RNA bases that hydrogen-bond with protein. SHAPE or fSHAPE coupled with enhanced crosslinking and immunoprecipitation (SHAPE-eCLIP or fSHAPE-eCLIP) pulls down RNAs bound by any protein of interest and returns their structure or protein interaction information, respectively. Here, we describe detailed protocols for SHAPE-eCLIP and fSHAPE-eCLIP and an analysis protocol for fSHAPE. For complete details on the use and execution of these protocols, please refer to Corley et al. (2020).

RNA Footprinting Using Small Chemical Reagents.

RNA is a pivotal element of the cell which is most of the time found in complex with protein(s) in a cellular environment. RNA can adopt three-dimensional structures that may form specific binding sites not only for proteins but for all sorts of molecules. Since the early days of molecular biology, strategies to probe RNA structure have been developed. Such probes are small molecules or RNases that most of the time specifically react with single strand nucleotides. The precise reaction or cleavage site can be mapped by reverse transcription. It appears that nucleotides in close contact or in proximity of a ligand are no longer reactive to these probes. Carrying the RNA probing experiment in parallel in presence and absence of a ligand yield differences that are known as the ligand "footprint." Such footprints allow for the identification of the precise site of the ligand interaction, but also reveals RNA structural rearrangement upon ligand binding. Here we provide an experimental and analytical workflow to carry RNA footprinting experiments.

Quantitative photoconversion analysis of internal molecular dynamics in stress granules and other membraneless organelles in live cells.

Photoconversion enables real-time labeling of protein sub-populations inside living cells, which can then be tracked with submicrometer resolution. Here, we detail the protocol of comparing protein dynamics inside membraneless organelles in live HEK293T cells using a CRISPR-Cas9 PABPC1-Dendra2 marker of stress granules. Measuring internal dynamics of membraneless organelles provides insight into their functional state, physical properties, and composition. Photoconversion has the advantage over other imaging techniques in that it is less phototoxic and allows for dual color tracking of proteins. For complete details on the use and execution of this protocol, please refer to Amen and Kaganovich (2020).

Micro RNA Sensing with Green Emitting Silver Nanoclusters.

Micro RNA (miR) are regulatory non-coding RNA molecules, which contain a small number of nucleotides ~18-28 nt. There are many various miR sequences found in plants and animals that perform important functions in developmental, metabolic, and disease processes. miRs can bind to complementary sequences within mRNA molecules thus silencing mRNA. Other functions include cardiovascular and neural development, stem cell differentiation, apoptosis, and tumors. In tumors, some miRs can function as oncogenes, others as tumor suppressors. Levels of certain miR molecules reflect cellular events, both normal and pathological. Therefore, miR molecules can be used as biomarkers for disease diagnosis and prognosis. One of these promising molecules is miR-21, which can serve as a biomarker with high potential for early diagnosis of various types of cancer. Here, we present a novel design of miR detection and demonstrate its efficacy on miR-21. The design employs emissive properties of DNA-silver nanoclusters (DNA/AgNC). The detection probe is designed as a hairpin DNA structure with one side of the stem complimentary to miR molecule. The binding of target miR-21 opens the hairpin structure, dramatically modulating emissive properties of AgNC hosted by the C12 loop of the hairpin. "Red" fluorescence of the DNA/AgNC probe is diminished in the presence of the target miR. At the same time, "green" fluorescence is activated and its intensity increases several-fold. The increase in intensity of "green" fluorescence is strong enough to detect the presence of miR-21. The intensity change follows the concentration dependence of the target miR present in a sample, which provides the basis of developing a new, simple probe for miR detection. The detection strategy is specific, as demonstrated using the response of the DNA/AgNC probe towards the scrambled miR-21 sequence and miR-25 molecule. Additionally, the design reported here is very sensitive with an estimated detection limit at ~1 picomole of miR-21.

Silver nanosol SERS quantitative analysis of ultratrace biotin coupled N-doped carbon dots catalytic amplification with affinity reaction.

Highly catalytic and stable N-doped carbon dots (N-CDs) were prepared rapidly by microwave procedure using glucose as precursor and ammonium sulfite as N-dopant. The reduction of AgNO3 by trisodium citrate (TCA) was slow to form nanosilver (AgNP), and the N-CDs exhibited strong catalysis of the AgNP reaction. The formed AgNPs were used as indicator in the presence of Vitoria blue B (VBB) molecule probe with a SERS peak at 1615 cm-1. With the increase of nancatalyst N-CDs concentration, the AgNP reaction speed up, and the SERS peak of VBB enhanced linearly due to formation of more AgNPs as substrate. In the presence of avidin (Ad), the SERS peak weakened. Upon addition of biotin, the SERS peak enhanced due to turn on the indicator nanoreaction. The enhanced SERS signal had a good linear relationship with the biotin concentration in range of 0.0006-0.021 ng/mL, with a detection limit of 0.3 pg/mL.

Glutathione-responsive disassembly of disulfide dicyanine for tumor imaging with reduction in background signal intensity.

Near-infrared (NIR) fluorescence imaging has been proved as an effective modality in identifying the tumor border and distinguishing the tumor cells from healthy tissue during the oncological surgery. Developing NIR fluorescent probes with high tumor to background (T/B) signal is essential for the complete debulking of the tumor, which will prolong the survival rate of tumor patients. However, the nonspecific binding and "always-on" properties of the conventional fluorescent probes leads to high background signals and poor specificity. Method: To address this problem, glutathione (GSH)-responsive, two disulfide-bonded dicyanine dyes (ss-diCy5 and ss-diNH800CW) were synthesized. As synthesized dyes are quenched under normal physiological conditions, however, once reached to the tumor site, these dyes are capable of emitting strong fluorescence signals primarily because of the cleavage of the disulfide bond in the tumor microenvironment with high GSH concentration. Besides, the GSH-responsive behavior of these dyes was monitored using the UV-vis and fluorescence spectroscopy. The diagnostic accuracy of the aforementioned dyes was also tested both in tumor cells and 4T1-bearing mice. Results: The fluorescence signal intensity of disulfide dicyanine dyes was quenched up to 89% compared to the mono cyanine dyes, thus providing a very low fluorescence background. However, when the disulfide dicyanine dye reaches the tumor site, the dicyanine is cleaved by GSH into two mono-dyes with high fluorescence strength, thus producing strong fluorescent signals upon excitation. The fluorescent signal of the dicyanine was enhanced by up to 27-fold after interacting with the GSH solution. In vivo xenografts tumor studies further revealed that the fluorescence signals of aforementioned dyes can be quickly recovered in the solid tumor. Conclusion: In summary, the disulfide dicyanines dyes can provide a promising platform for specific tumor-activatable fluorescence imaging with improved T/B value.

Enhanced Probe-Based RT-qPCR Quantification of MicroRNAs Using Poly(A) Tailing and 5' Adaptor Ligation.

Probe-based quantitative PCR (qPCR) is a commonly used tool in the realm of real-time qPCR experiments since it is one of the most sensitive detection methods allowing an accurate and reproducible analysis. It uses real-time fluorescence from a fluorescently labeled probe that specifically targets the desired PCR product to measure DNA amplification at each cycle of the PCR. Coupled to a proper reverse transcription step, probe-based qPCR can be efficiently used for the analysis of the expression of difficult targets such as miRNAs. In this chapter, we describe the TaqMan® advanced miRNA assay in which, owing to a poly(A)-tailing step, the reverse transcription is advantageously performed at once for all the miRNAs in a given sample, and, coupled to the ligation of a 5' universal adapter, allows for a supplementary pre-qPCR amplification step increasing the sensitivity of the assay. Along this protocol, we also provide our general guidelines and advices to perform a reliable and successful quantitative analysis.

DNA methylation assay using droplet-based DNA melting curve analysis.

DNA methylation is an epigenetic regulation of gene expression, which has drawn great attention in biomedical research due to its association with various diseases. A robust, inexpensive platform to detect and quantify the methylation status in a specific genomic region is necessary. In this study, an on-chip analytical technique of cytosine methylation with droplets in a microchannel is proposed. Genomic DNA samples are encapsulated into a series of droplets and transported through a detection region, where a stable temperature gradient is created. As the temperature is elevated from 60 °C to 85 °C, the DNA samples denature and the associated fluorescence signals decay, with the relationship being acquired as the melting curve. The droplets serve as discrete reactors for conducting DNA melting curve analysis in the liquid phase, thereby eliminating the need for immobilization of reagents. Due to a high heating rate and greater enhanced thermal stability, this microchip allows larger melting temperature differences for the samples at different percentages of methylated DNA. It has an enhanced discrimination ability and lower volume consumption, compared to the commercial qPCR machine. This chip enables quantification of the methylation levels of the pluripotent stem cell factor Oct-4 in its distal enhancer (DE) region, with a designed probe after bisulfite treatment and asymmetric PCR.

Implantation of Chronic Silicon Probes and Recording of Hippocampal Place Cells in an Enriched Treadmill Apparatus.

An important requisite for understanding brain function is the identification of behavior and cell activity correlates. Silicon probes are advanced electrodes for large-scale electrophysiological recording of neuronal activity, but the procedures for their chronic implantation are still underdeveloped. The activity of hippocampal place cells is known to correlate with an animal's position in the environment, but the underlying mechanisms are still unclear. To investigate place cells, here we describe a set of techniques which range from the fabrication of devices for chronic silicon probe implants to the monitoring of place field activity in a cue-enriched treadmill apparatus. A micro-drive and a hat are built by fitting and fastening together 3D-printed plastic parts. A silicon probe is mounted on the micro-drive, cleaned, and coated with dye. A first surgery is performed to fix the hat on the skull of a mouse. Small landmarks are fabricated and attached to the belt of a treadmill. The mouse is trained to run head-fixed on the treadmill. A second surgery is performed to implant the silicon probe in the hippocampus, following which broadband electrophysiological signals are recorded. Finally, the silicon probe is recovered and cleaned for reuse. The analysis of place cell activity in the treadmill reveals a diversity of place field mechanisms, outlining the benefit of the approach.

Label-free thioflavin T/G-quadruplex-based real-time strand displacement amplification for biosensing applications.

To promote application of strand-displacement amplification (SDA) techniques in biosensing, a label-free, real-time monitoring strategy for isothermal nucleic acid amplification reactions was designed. G-quadruplex structures were introduced into SDA products using specific recognition of G-quadruplexes by the fluorogenic dye thioflavin T. Performance was good for real-time monitoring of traditional SDA by a linear-amplification mechanism and for exponential cross-triggered SDA amplification. The strategy worked on a commercial real-time PCR instrument, making it suitable for biosensing platforms. As examples, two highly sensitive and specific biosensors were designed for analysis of the activity of uracil-DNA glycosylase (UDG) and the restriction endonuclease EcoRI. Detection limits were 6×10(-5)U/mL for UDG and 0.016U/mL for EcoRI. Detection of corresponding targets in complex matrices such as cell lysates or human serum was also demonstrated. Compared to traditional end-point detection methods, real-time SDA-based approaches have the advantages of simple, fast operation; high sensitivity; low risk of carryover contamination; and very high throughput. The introduction of real-time monitoring strategies may promote application of SDA reactions in biosensor design.

A G-quadruplex-selective luminescent probe with an anchor tail for the switch-on detection of thymine DNA glycosylase activity.

Thymine DNA glycosylase (TDG) performs essential functions in maintaining genetic integrity and epigenetic regulation, which also plays an essential role in DNA demethylation. In this work, the novel iridium(III) complex 1 with an anchor tail was synthesized and employed to construct a G-quadruplex-based assay for detecting TDG activity in aqueous solution by using the mismatched base excising property of TDG with T4 DNA ligase and phi29 DNA polymerase, in concert with the rolling circle amplification (RCA) strategy. The assay achieved a detection limit of 0.048UmL(-)(1) (0.012ngmL(-1)), and showed high selectivity towards TDG even in the presence of other proteins and enzymes. Additionally, the assay could function in diluted cellular debris.

Electrochemical detection of Nanog in cell extracts via target-induced resolution of an electrode-bound DNA pseudoknot.

Nanog is among the most important indicators of cell pluripotency and self-renew, so detection of Nanog is critical for tumor assessment and monitoring of clinical prognosis. In this work, a novel method for Nanog detection is proposed by using electrochemical technique based on target-induced conformational change of an electrode-bound DNA pseudoknot. In the absence of Nanog, the rigid structure of the pseudoknot will minimize the connection between the redox tag and the electrode, thus reducing the obtained faradaic current. Nevertheless, the Nanog binding may liberate the flexible single-stranded element that transforms the DNA pesudokont into DNA hairpin structure due to steric hindrance effect, thus making the electrochemical tag close to the electrode surface. Consequently, electron transfer can be enhanced and very well electrochemical response can be observed. By using the proposed method, Nanog can be determined in a linear range from 2nM to 25nM with a detection limit of 163 pM. Furthermore, the proposed method can be directly used to assay Nanog not only in purified samples but also in complex media (cell extracts), which shows potential applications in Nanog functional studies as well as clinical diagnosis in the future.

Ultrasensitive, colorimetric detection of microRNAs based on isothermal exponential amplification reaction-assisted gold nanoparticle amplification.

MicroRNAs (miRNAs) play important roles in a wide range of biological processes, and their aberrant expressions are linked to a large number of human diseases and disorders. In this work, we developed a colorimetric method for rapid, ultrasensitive miRNA detection via isothermal exponential amplification reaction (EXPAR)-assisted gold nanoparticle (AuNP) amplification. The sensing probe designed with a tandem phosphorothioate modification in the backbone of the polyadenines at the 5' terminus was employed to directly assemble onto the surface of AuNP with high adsorption affinity. The recognition domain at the 3' terminus of the sensing probe hybridizes with target miRNAs to trigger EXPAR with exponential signal amplification. With the amplification reaction with the action of DNA polymerase, the sensing probe gradually detaches from the AuNP, resulting in the aggregation of bare AuNPs in the high-salt reaction environment due to lack of DNA protection. The presence of AuNP aggregation is conveniently measured by UV-vis spectroscopy. Our proposed method could provide a linear detection range from 50fM to 10nM with a detection limit of ∼46fM within 60min, and also discriminate a single-nucleotide difference between homologous miRNAs.

Discovering the enzyme mimetic activity of metal-organic framework (MOF) for label-free and colorimetric sensing of biomolecules.

A label-free sensing strategy based on the enzyme-mimicking activity of MOF was demonstrated for colorimetric detection of biomolecules. Firstly obvious blue color was observed due to the high efficiency of peroxidase-like catalytic activity of Fe-MIL-88A (an ion-based MOF material) toward 3,3',5,5'-tetramethylbenzidine (TMB). Then in the presence of target biomolecule and corresponding aptamer, the mimetic activity of Fe-MIL-88A can be strongly inhibited and used directly to realize the colorimetric detection. On the basis of the interesting findings, we designed a straightforward, label-free and sensitive colorimetric method for biomolecule detection by using the enzyme mimetic property of MOF coupling with molecular recognition element. Compared with the existed publications, our work breaks the routine way by setting up an inorganic-organic MOF-aptamer hybrid platform for colorimetric determination of biomolecules, expanding the targets scope from H2O2 or glucose to biomolecules. As a proof of concept, thrombin and thrombin aptamer was used as a model analyte. The limit of detection of 10nM can be achieved with naked eyes and ultrahigh selectivity of thrombin toward numerous interfering substances with 10-fold concentration was demonstrated significantly. Of note, the method was further applied for the detection of thrombin in human serum samples, showing the results in agreement with those values obtained in an immobilization buffer by the colorimetric method. This inorganic-organic MOF-aptamer sensing strategy may in principle be universally applicable for the detection of a range of environmental or biomedical molecules of interests.

Sensitive detection of multiple pathogens using a single DNA probe.

A simple but promising electrochemical DNA nanosensor was designed, constructed and applied to differentiate a few food-borne pathogens. The DNA probe was initially designed to have a complementary region in Vibrio parahaemolyticus (VP) genome and to make different hybridization patterns with other selected pathogens. The sensor was based on a screen printed carbon electrode (SPCE) modified with polylactide-stabilized gold nanoparticles (PLA-AuNPs) and methylene blue (MB) was employed as the redox indicator binding better to single-stranded DNA. The immobilization and hybridization events were assessed using differential pulse voltammetry (DPV). The fabricated biosensor was able to specifically distinguish complementary, non-complementary and mismatched oligonucleotides. DNA was measured in the range of 2.0×10(-9)-2.0×10(-13)M with a detection limit of 5.3×10(-12)M. The relative standard deviation for 6 replications of DPV measurement of 0.2µM complementary DNA was 4.88%. The fabricated DNA biosensor was considered stable and portable as indicated by a recovery of more than 80% after a storage period of 6 months at 4-45°C. Cross-reactivity studies against various food-borne pathogens showed a reliably sensitive detection of VP.

Biomimetic nanochannels based biosensor for ultrasensitive and label-free detection of nucleic acids.

A very simple sensing device based on biomimetic nanochannels has been developed for label-free, ultrasensitive and highly sequence-specific detection of DNA. Probe DNA was modified on the inner wall of the nanochannel surface by layer-by-layer (LBL) assembly. After probe DNA immobilization, DNA detection was realized by monitoring the rectified ion current when hybridization occurred. Due to three dimensional (3D) nanoscale environment of the nanochannel, this special geometry dramatically increased the surface area of the nanochannel for immobilization of probe molecules on the inner-surface and enlarged contact area between probes and target-molecules. Thus, the unique sensor reached a reliable detection limit of 10 fM for target DNA. In addition, this DNA sensor could discriminate complementary DNA (c-DNA) from non-complementary DNA (nc-DNA), two-base mismatched DNA (2bm-DNA) and one-base mismatched DNA (1bm-DNA) with high specificity. Moreover, the nanochannel-based biosensor was also able to detect target DNA even in an interfering environment and serum samples. This approach will provide a novel biosensing platform for detection and discrimination of disease-related molecular targets and unknown sequence DNA.

Simultaneous and multiplexed detection of exosome microRNAs using molecular beacons.

Simultaneous and multiplexed detection of microRNAs (miRNAs) in a whole exosome is developed, which can be utilized as a PCR-free efficient diagnosis method for various diseases. Exosomes are small extracellular vesicles that contain biomarker miRNAs from parental cells. Because they circulate throughout bodily fluids, exosomal biomarkers offer great advantages for diagnosis in many aspects. In general, PCR-based methods can be used for exosomal miRNA detection but they are laborious, expensive, and time-consuming, which make them unsuitable for high-throughput diagnosis of diseases. Previously, we reported that single miRNA in the exosomes can be detected specifically using an oligonucleotide probe or molecular beacon. Herein, we demonstrate for the first time that multiple miRNAs can be detected simultaneously in exosomes using miRNA-targeting molecular beacons. Exosomes from a breast cancer cell line, MCF-7, were used for the production of exosomes because MCF-7 has a high level of miR-21, miR-375, and miR-27a as target miRNAs. Molecular beacons successfully hybridized with multiple miRNAs in the cancer cell-derived exosomes even in the presence of high human serum concentration. In addition, it is noteworthy that the choice of fluorophores for multiplexing biomarkers in an exosome is crucial because of its small size. The proposed method described in this article is beneficial to high-throughput analysis for disease diagnosis, prognosis, and response to treatment because it is a time-, labor-, and cost-saving technique.

A mini-review on functional nucleic acids-based heavy metal ion detection.

Recent years have witnessed great progress in developing functional nucleic acids (FNAs)-based sensors for the detection of heavy metal ion. In this review, four types of the FNAs that most widely-used in heavy metal ions detection were briefly introduced and a dozen of recently published review articles which summarized those FNAs-based sensors were introduced. Particularly, according to the degree of automation and system integration, those FNAs-based sensors which belong to the lab-on-a-chip (LOC) category were reviewed in more detail by classifying them into six types such as microfluidic LOC system, microchip, lateral flow dipstick, personal glucose meter, microfluidic paper-based analytical devices (µPADs) and disc-based analytical platform. After gave a brief description of the sensing strategies, properties, advantages or disadvantages of these FNAs-based sensors, existing problems and future perspectives were also discussed.

Magnetic field-assisted SPR biosensor based on carboxyl-functionalized graphene oxide sensing film and Fe3O4-hollow gold nanohybrids probe.

A novel surface plasmon resonance (SPR) biosensor, coupled with the magnetic bioseparation technique, was constructed and used to the determination of human IgG. Carboxyl-functionalized graphene oxide (cGO) sheet was employed as the sensing film for the efficient immobilization of capture antibody (Ab1). Nanoconjugates (FHAb2), obtained by binding detection antibody (Ab2) to the nanohybrids containing Fe3O4 nanoparticles (Fe3O4 NPs) and hollow gold sphere nanoparticles (HGNPs), were used to specifically collect the target analytes from sample solutions and serve as labels. Owing to the notable plasmonic fields spreading over inner and outer surfaces, HGNPs played key roles in amplifying the SPR response signals originating from the dielectric changes on the sensing films during the binding of Ab1 and human IgG-Ab2FH complexes. In addition, FHAb2 were also used as "vehicles" for the rapid delivery of the separated and enriched target analytes from sample solutions to the sensor surface via an external magnet. In the present method, taking advantages of the magnetic field-driven mass transfer and the significant signal amplification effect of FHAb2, the separation and analysis of human IgG in serum samples are quite effective and sensitive. The limit of detection was 1.88ngmL(-1), which is about 260-fold lower than that obtained by routine SPR biosensors with sandwich assay.

An ultrasensitive SiO2-encapsulated alloyed CdZnSeS quantum dot-molecular beacon nanobiosensor for norovirus.

Ultrasensitive, rapid and selective diagnostic probes are urgently needed to overcome the limitations of traditional probes for norovirus (NV). Here, we report the detection of NV genogroup II via nucleic acid hybridization technology using a quantum dot (QD)-conjugated molecular beacon (MB) probe. To boost the sensitivity of the MB assay system, an ultrasensitive QD fluorophore with unique optical properties was synthesized, characterized and exploited as a fluorescence signal generator. Alloyed thioglycolic (TGA)-capped CdZnSeS QDs with a high photoluminescence (PL) quantum yield (QY) value of 92% were synthesized, and a modified silanization method was employed to encapsulate the thiol-capped QDs in a silica layer. The resulting highly luminescent alloyed SiO2-coated CdZnSeS QDs had a remarkable PL QY value of 98%. Transmission electron microscopy and dynamic light scattering confirmed the monodispersity of the alloyed nanocrystals, and zeta potential analysis confirmed their colloidal stability. Powder X-ray diffraction and PL lifetime measurements confirmed the surface modification of the QDs. The alloyed TGA-capped and SiO2-coated CdZnSeS QD-conjugated MB bioprobes detected extremely low concentrations of NV RNA. Ultrasensitive detection of low concentrations of NV RNA with a limit of detection (LOD) of 8.2copies/mL in human serum and a LOD of 9.3 copies/mL in buffer was achieved using the SiO2-coated CdZnSeS QD-MB probes, an increase in sensitivity of 3-fold compared with the detection limit for NV RNA using TGA-capped CdZnSeS QD-MBs. The additional merits of our detection system are rapidity, specificity and improved sensitivity over conventional molecular test probes.