Probing G-quadruplex topologies and recognition concurrently in real time and 3D using a dual-app nucleoside probe.

Comprehensive understanding of structure and recognition properties of regulatory nucleic acid elements in real time and atomic level is highly important to devise efficient therapeutic strategies. Here, we report the establishment of an innovative biophysical platform using a dual-app nucleoside analog, which serves as a common probe to detect and correlate different GQ structures and ligand binding under equilibrium conditions and in 3D by fluorescence and X-ray crystallography techniques. The probe (SedU) is composed of a microenvironment-sensitive fluorophore and an excellent anomalous X-ray scatterer (Se), which is assembled by attaching a selenophene ring at 5-position of 2'-deoxyuridine. SedU incorporated into the loop region of human telomeric DNA repeat fluorescently distinguished subtle differences in GQ topologies and enabled quantify ligand binding to different topologies. Importantly, anomalous X-ray dispersion signal from Se could be used to determine the structure of GQs. As the probe is minimally perturbing, a direct comparison of fluorescence data and crystal structures provided structural insights on how the probe senses different GQ conformations without affecting the native fold. Taken together, our dual-app probe represents a new class of tool that opens up new experimental strategies to concurrently investigate nucleic acid structure and recognition in real time and 3D.

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.

A comprehensive model for the diffusion and hybridization processes of nucleic acid probes in fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) has been extensively used in the past decades for the detection and localization of microorganisms. However, a mechanistic approach of the whole FISH process is still missing, and the main limiting steps for the hybridization to occur remain unclear. In here, FISH is approached as a particular case of a diffusion-reaction kinetics, where molecular probes (MPs) move from the hybridization solution to the target RNA site within the cells. Based on literature models, the characteristic times taken by different MPs to diffuse across multiple cellular barriers, as well as the reaction time associated with the formation of the duplex molecular probe-RNA, were estimated. Structural and size differences at the membrane level of bacterial and animal cells were considered. For bacterial cells, the limiting step for diffusion is likely to be the peptidoglycan layer (characteristic time of 7.94 × 102 - 4.39 × 103 s), whereas for animal cells, the limiting step should be the diffusion of the probe through the bulk (1.8-5.0 s) followed by the diffusion through the lipid membrane (1 s). The information provided here may serve as a basis for a more rational development of FISH protocols in the future.

Visual genotyping of thalassemia by using pyrrolidinyl peptide nucleic acid probes immobilized on carboxymethylcellulose-modified paper and enzyme-induced pigmentation.

A simple probe pair was designed for the detection of hemoglobin E (HbE) genotype, a single-point mutation that leads to abnormal red blood cells commonly found in South East Asia. The key to differentiation is the use of a conformationally constrained peptide nucleic acid (PNA) that was immobilized on carboxymethylcellulose-modified paper. This was then used for target DNA binding and visualization by an enzyme-catalyzed pigmentation. The biotinylated target DNA bound to the immobilized probe was visually detected via alkaline phosphatase-linked streptavidin. This enzyme conjugate catalyzed the dephosphorylation of the substrate 5-bromo-4-chloro-3-indolyl phosphate, leading to a series of reactions that generate an intense, dark blue pigment. The test was validated with 100 DNA samples, which shows good discrimination among different genotypes (normal, HbE, and heterozygous) with 100% accuracy when optimal conditions of analysis were applied. The method does not require temperature control and can be performed at ambient temperature. This is an attractive feature for diagnosis in primary care, which accounts for a large part of affected population. Graphical abstract Schematic representation of a paper-based sensor for the detection of the gene Hemoglobin E. The interaction between an immobilized peptide nucleic acid and a DNA target leads to enzymatic pigmentation, allowing simple visual readout with up to 100% accuracy.

RiboCAT: a new capillary electrophoresis data analysis tool for nucleic acid probing.

Chemical and enzymatic probing of RNA secondary structure and RNA/protein interactions provides the basis for understanding the functions of structured RNAs. However, the ability to rapidly perform such experiments using capillary electrophoresis has been hampered by relatively labor-intensive data analysis software. While these computationally robust programs have been shown to calculate residue-specific reactivities to a high degree of accuracy, they often require time-consuming manual intervention and lack the ability to be easily modified by users. To alleviate these issues, RiboCAT (Ribonucleic acid capillary-electrophoresis analysis tool) was developed as a user-friendly, Microsoft Excel-based tool that reduces the need for manual intervention, thereby significantly shortening the time required for data analysis. Features of this tool include (i) the use of an Excel platform, (ii) a method of intercapillary signal alignment using internal size standards, (iii) a peak-sharpening algorithm to more accurately identify peaks, and (iv) an open architecture allowing for simple user intervention. Furthermore, a complementary tool, RiboDOG (RiboCAT data output generator) was designed to facilitate the comparison of multiple data sets, highlighting potential inconsistencies and inaccuracies that may have occurred during analysis. Using these new tools, the secondary structure of the HIV-1 5' untranslated region (5'UTR) was determined using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE), matching the results of previous work.

Paper-based point-of-care test with xeno nucleic acid probes.

Bridging the unmet need of efficient point-of-care testing (POCT) in biomedical engineering research and practice with the emerging development in artificial synthetic xeno nucleic acids (XNAs), this review summarized the recent development in paper-based POCT using XNAs as sensing probes. Alongside the signal transducing mode and immobilization methods of XNA probes, a detailed evaluation of probe performance was disclosed. With these new aspects, both researchers in synthetic chemistry / biomedical engineering and physicians in clinical practice could gain new insights in designing, manufacturing and choosing suitable reagents and techniques for POCT.

Nanoscale Nucleic Acid Recognition at the Solid-Liquid Interface Using Xeno Nucleic Acid Probes.

Challenges in reliable nucleic acid detection are manifold. The major ones are related to false positive or negative signals due to a lack of target specificity in detection and to low sensitivity, especially when a plethora of background sequences are present that can mask the specific recognition signal. Utilizing designed synthetic nucleic acids that are commonly called xeno nucleic acids could offer potential routes to meeting such challenges. In this article, we present the general framework of nucleic acid detection, especially for nanoscale applications, and discuss how and why the xeno nucleic acids could be truly an alternative to the DNA probes. Two specific cases, locked nucleic acid (LNA) and peptide nucleic acid (PNA), which are nuclease-resistant and can form thermally stable duplexes with DNA, are addressed. It is shown that the relative ease of the conformationally rigid LNA probe to be oriented upright on the substrate surface and of the nonionic PNA probe to result into high probe density assists in their use in nanoscale nucleic acid recognition. It is anticipated that success with these probes may lead to important developments such as PCR-independent approaches where the major aim is to detect a small number of target sequences present in the analyte medium.

A universal aptasensing platform based on cryonase-assisted signal amplification and graphene oxide induced quenching of the fluorescence of labeled nucleic acid probes: application to the detection of theophylline and ATP.

This study describes a universal fluorometric method for sensitive detection of analytes by using aptamers. It is based on the use of graphene oxide (GO) and cryonase-assisted signal amplification. GO is a strong quencher of FAM-labeled nucleic acid probes, while cryonase digests all types of nucleic acid probes. This makes the platform widely applicable to analytes for which the corresponding aptamers are available. Theophylline and ATP were chosen as model analytes. In the absence of targets, dye-labeled aptamers are in a flexible single strand state and adsorb on the GO. As a result, the probes are non-fluorescent due to the efficient quenching of dyes by GO. Upon the addition of a specific target, the aptamer/target complex desorbed from the GO surface and the probe becomes fluorescent. The released complex will immediately become a substrate for cryonase digestion and subsequently releasing the target to bind to another aptamer to initiate the next round of cleavage. This cyclic reaction will repeat again and again until all the related-probes are consumed and all fluorophores light up, resulting in significant fluorescent signal amplification. The detection limits are 47 nM for theophylline and 22.5 nM for ATP. This is much better than that of known methods. The assay requires only mix-and-measure steps that can be accomplished rapidly. In our perception, the detection scheme holds great promise for the design enzyme-aided amplification mechanisms for use in bioanalytical methods. Graphical abstract A cryonase-assisted signal amplification (CASA) method has been developed by using graphene oxide (GO) conjugated with a fluorophore-labeled aptamer for fluorescence signal generation. It has a large scope because it may be applied to numerous analytes.

Continuously tunable nucleic acid hybridization probes.

In silico-designed nucleic acid probes and primers often do not achieve favorable specificity and sensitivity tradeoffs on the first try, and iterative empirical sequence-based optimization is needed, particularly in multiplexed assays. We present a novel, on-the-fly method of tuning probe affinity and selectivity by adjusting the stoichiometry of auxiliary species, which allows for independent and decoupled adjustment of the hybridization yield for different probes in multiplexed assays. Using this method, we achieved near-continuous tuning of probe effective free energy. To demonstrate our approach, we enforced uniform capture efficiency of 31 DNA molecules (GC content, 0-100%), maximized the signal difference for 11 pairs of single-nucleotide variants and performed tunable hybrid capture of mRNA from total RNA. Using the Nanostring nCounter platform, we applied stoichiometric tuning to simultaneously adjust yields for a 24-plex assay, and we show multiplexed quantitation of RNA sequences and variants from formalin-fixed, paraffin-embedded samples.

Poly-adenine-mediated fluorescent spherical nucleic acid probes for live-cell imaging of endogenous tumor-related mRNA.

Identification of tumor-related mRNA in living cells hold great promise for early cancer diagnosis and pathological research. Herein, we present poly-adenine (polyA)-mediated fluorescent spherical nucleic acid (FSNA) probes for intracellular mRNA detection with regulable sensitivities by programmably adjusting the loading density of DNA on gold nano-interface. Gold nanoparticles (AuNPs) functionalized with polyA-tailed recognition sequences were hybridized to fluorescent "reporter" strands to fabricate fluorescence-quenched FSNA probes. While exposed to target gene, the "reporter" strands were released from FSNA through strand displacement and fluorescence was recovered. With polyA20 tail as the attaching block, the detection limit of FSNA probes was calculated to be 0.31 nM, which is ~55 fold lower than that of thiolated probes without surface density regulation. Quantitative intracellular mRNA detection and imaging could be achieved with polyA-mediated FSNA probes within 2 hours, indicating their application potential in rapid and sensitive intracellular target imaging.

Palladium-Mediated Labeling of Nucleic Acids.

New applications of Pd-catalyzed coupling reactions (Suzuki-Miyaura, Sonogashira, and Stille-Migita coupling) for post-conjugation of nucleic acids have been developed recently. Breakthroughs in this area might now pave the way for the development of sophisticated DNA probes, which might be of great interest in chemical biology, nanotechnology, and bioanalysis, as well as in diagnostic domains.

Probing Human Telomeric DNA and RNA Topology and Ligand Binding in a Cellular Model by Using Responsive Fluorescent Nucleoside Probes.

The development of biophysical systems that enable an understanding of the structure and ligand-binding properties of G-quadruplex (GQ)-forming nucleic acid sequences in cells or models that mimic the cellular environment would be highly beneficial in advancing GQ-directed therapeutic strategies. Herein, the establishment of a biophysical platform to investigate the structure and recognition properties of human telomeric (H-Telo) DNA and RNA repeats in a cell-like confined environment by using conformation-sensitive fluorescent nucleoside probes and a widely used cellular model, bis(2-ethylhexyl) sodium sulfosuccinate reverse micelles (RMs), is described. The 2'-deoxy and ribonucleoside probes, composed of a 5-benzofuran uracil base analogue, faithfully report the aqueous micellar core through changes in their fluorescence properties. The nucleoside probes incorporated into different loops of H-Telo DNA and RNA oligonucleotide repeats are minimally perturbing and photophysically signal the formation of respective GQ structures in both aqueous buffer and RMs. Furthermore, these sensors enable a direct comparison of the binding affinity of a ligand to H-Telo DNA and RNA GQ structures in the bulk and confined environment of RMs. These results demonstrate that this combination of a GQ nucleoside probe and easy-to-handle RMs could provide new opportunities to study and devise screening-compatible assays in a cell-like environment to discover GQ binders of clinical potential.

Amplification-Free Detection of Circulating microRNA Biomarkers from Body Fluids Based on Fluorogenic Oligonucleotide-Templated Reaction between Engineered Peptide Nucleic Acid Probes: Application to Prostate Cancer Diagnosis.

Highly abundant in cells, microRNAs (or miRs) play a key role as regulators of gene expression. A proportion of them are also detectable in biofluids making them ideal noninvasive biomarkers for pathologies in which miR levels are aberrantly expressed, such as cancer. Peptide nucleic acids (PNAs) are engineered uncharged oligonucleotide analogues capable of hybridizing to complementary nucleic acids with high affinity and high specificity. Herein, novel PNA-based fluorogenic biosensors have been designed and synthesized that target miR biomarkers for prostate cancer (PCa). The sensing strategy is based on oligonucleotide-templated reactions where the only miR of interest serves as a matrix to catalyze an otherwise highly unfavorable fluorogenic reaction. Validated in vitro using synthetic RNAs, these newly developed biosensors were then shown to detect endogenous concentrations of miR in human blood samples without the need for any amplification step and with minimal sample processing. This low-cost, quantitative, and versatile sensing technology has been technically validated using gold-standard RT-qPCR. Compared to RT-qPCR however, this enzyme-free, isothermal blood test is amenable to incorporation into low-cost portable devices and could therefore be suitable for widespread public screening.

Oxo-Functionalized Graphene as a Cell Membrane Carrier of Nucleic Acid Probes Controlled by Aging.

We applied a fluorescein-containing oligonucleotide (ON) to probe surface properties of oxidized graphene (oxo-G) and observed that graphene-like patches are formed upon aging of oxo-G, indicated by enhanced probe binding and by FTIR spectroscopic analysis. By using a recently developed fluorogenic endoperoxide (EP) probe, we confirmed that during the aging process the amount of EPs on the oxo-G surface is reduced. Furthermore, aging was found to strongly affect cell membrane carrier properties of this material. In particular, freshly prepared oxo-G does not act as a carrier, whereas oxo-G aged for 28 days at 4 °C is an excellent carrier. Based on these data we prepared an optimized oxo-G, which has a low-defect density, binds ONs, is not toxic, and acts as cell membrane carrier. We successfully applied this material to design fluorogenic probes of representative intracellular nucleic acids 28S rRNA and ß-actin-mRNA. The results will help to standardize oxidized graphene derivatives for biomedical and bioanalytical applications.

Discriminating unalike single nucleobase mismatches using a molecularly resolved, label-free, interfacial LNA-based assay.

A number of reports have been made in recent times on label-free detection of nucleic acid sequences. However, most of these studies deal with ensemble measurements, therefore lacking in molecular level resolution. These assays have usually employed ssDNA sensor probes, and often suffered from problems of irreproducibility and poor sequence-selectivity. Herein, the applicability of surface-anchored single stranded locked nucleic acid (ssLNA) probes has been assessed in the detection of target DNA sequences, as an alternative to the DNA-based assay. Importantly, the effectiveness of the LNA-based assay in identifying different types of single nucleobase mismatches has been tested. Since the duplex melting temperature is an indicator of duplex stability, the ensemble on-surface Tm values of the surface-confined LNA-DNA duplexes have been compared to the duplex unbinding force values obtained from atomic force spectroscopy (AFS) experiments. A common mismatch discrimination pattern elicited by both the ensemble and the molecular level AFS approach could be identified. Apart from quantitative delineation of the different types of mismatches, the label-free AFS analysis confirms different degrees of efficiency of the purine and pyrimidine bases, present on the LNA backbone, in discriminating different nucleobase mismatch types. Importantly, the LNA-based AFS analysis can distinguish between the disease-relevant gene fragments, e.g., multidrug-resistant Mycobacterium tuberculosis (MTB) mutation, and the wild type. Since LNA probes are nuclease-resistant, these findings could potentially pave way to diagnostic applications of the LNA-based AFS assay.

A NASBA on microgel-tethered molecular-beacon microarray for real-time microbial molecular diagnostics.

Despite their many advantages and successes, molecular beacon (MB) hybridization probes have not been extensively used in microarray formats because of the complicating probe-substrate interactions that increase the background intensity. We have previously shown that tethering to surface-patterned microgels is an effective means for localizing MB probes to specific surface locations in a microarray format while simultaneously maintaining them in as water-like an environment as possible and minimizing probe-surface interactions. Here we extend this approach to include both real-time detection together with integrated NASBA amplification. We fabricate small (∼250 µm × 250 µm) simplex, duplex, and five-plex assays with microarray spots of controllable size (∼20 µm diameter), position, and shape to detect bacteria and fungi in a bloodstream-infection model. The targets, primers, and microgel-tethered probes can be combined in a single isothermal reaction chamber with no post-amplification labelling. We extract total RNA from clinical blood samples and differentiate between Gram-positive and Gram-negative bloodstream infection in a duplex assay to detect RNA- amplicons. The sensitivity based on our current protocols in a simplex assay to detect specific ribosomal RNA sequences within total RNA extracted from S. aureus and E. coli cultures corresponds to tens of bacteria per ml. We furthermore show that the platform can detect RNA- amplicons from synthetic target DNA with 1 fM sensitivity in sample volumes that contain about 12 000 DNA molecules. These experiments demonstrate an alternative approach that can enable rapid and real-time microarray-based molecular diagnostics.

In Vitro Reversible Translation Control Using γPNA Probes.

On-demand regulation of gene expression in living cells is a central goal of chemical biology and antisense therapeutic development. While significant advances have allowed regulatory modulation through inserted genetic elements, on-demand control of the expression/translation state of a given native gene by complementary sequence interactions remains a technical challenge. Toward this objective, we demonstrate the reversible suppression of a luciferase gene in cell-free translation using Watson-Crick base pairing between the mRNA and a complementary γ-modified peptide nucleic acid (γPNA) sequence with a noncomplementary toehold. Exploiting the favorable thermodynamics of γPNA-γPNA interactions, the antisense sequence can be removed by hybridization of a second, fully complementary γPNA, through a strand displacement reaction, allowing translation to proceed. Complementary RNA is also shown to displace the bound antisense γPNA, opening up possibilities of in vivo regulation by native gene expression.

Design, Preparation, and Characterization of PNA-Based Hybridization Probes for Affibody-Molecule-Mediated Pretargeting.

In radioimmunotherapy, the contrast between tumor and normal tissue can be improved by using a pretargeting strategy with a primary targeting agent, which is conjugated to a recognition tag, and a secondary radiolabeled molecule binding specifically to the recognition tag. The secondary molecule is injected after the targeting agent has accumulated in the tumor and is designed to have a favorable biodistribution profile, with fast clearance from blood and low uptake in normal tissues. In this study, we have designed and evaluated two complementary peptide nucleic acid (PNA)-based probes for specific and high-affinity association in vivo. An anti-HER2 Affibody-PNA chimera, Z(HER2:342)-SR-HP1, was produced by a semisynthetic approach using sortase A catalyzed ligation of a recombinantly produced Affibody molecule to a PNA-based HP1-probe assembled using solid-phase chemistry. A complementary HP2 probe carrying a DOTA chelator and a tyrosine for dual radiolabeling was prepared by solid-phase synthesis. Circular dichroism (CD) spectroscopy and UV thermal melts showed that the probes can hybridize to form a structured duplex with a very high melting temperature (T(m)), both in HP1:HP2 and in Z(HER2:342)-SR-HP1:HP2 (T(m) = 86-88 °C), and the high binding affinity between Z(HER2:342)-SR-HP1 and HP2 was confirmed in a surface plasmon resonance (SPR)-based binding study. Following a moderately fast association (1.7 × 10(5) M(-1) s(-1)), the dissociation of the probes was extremely slow and <5% dissociation was observed after 17 h. The equilibrium dissociation constant (K(D)) for Z(HER2:342)-SR-HP1:HP2 binding to HER2 was estimated by SPR to be 212 pM, suggesting that the conjugation to PNA does not impair Affibody binding to HER2. The biodistribution profiles of (111)In- and (125)I-labeled HP2 were measured in NMRI mice, showing very fast blood clearance rates and low accumulation of radioactivity in kidneys and other organs. The measured radioactivity in blood was 0.63 ± 0.15 and 0.41 ± 0.15%ID/g for (125)I- and (111)In-HP2, respectively, at 1 h p.i., and at 4 h p.i., the kidney accumulation of radioactivity was 0.17 ± 0.04%ID/g for (125)I-HP2 and 3.83 ± 0.39%ID/g for (111)In-HP2. Taken together, the results suggest that a PNA-based system has suitable biophysical and in vivo properties and is a promising approach for pretargeting of Affibody molecules.

Hybrid, multiplexed, functional DNA nanotechnology for bioanalysis.

We herein aim to report on the fabrication of DNA nano-heterostructures usable as a robust multi-functional analytical system to obtain multiple and complex data in parallel format from a single sample with unprecedented analytical performances. The ability of chemical information contained in the sequences of programmed DNA structures to organize matter made DNA become a unique material in "the nanoworld". Such carefully designed DNA nanostructures can then be functionalized/templated with different biomolecules/nanomaterials as different as nanoparticles, nanowires, organic molecules, peptides, and proteins with controlled spacing on the nanometer scale (<10 nm). In this way, it is possible to combine the properties of both DNA and nanomaterials for exposing the designed functionality and customizable geometrical hetero-nanostructures. By coupling automated on-chip high yield DNA synthesis with low cost detection methods, DNA-nanotechnology can enable the realization of high-sensitivity, multiplexed bioanalytical assays for many different applications like diagnostics, drug screening, toxicology, immunology and biosensors.

Fluorescence monitoring of riboswitch transcription regulation using a dual molecular beacon assay.

Riboswitches are mRNA elements that specifically bind cellular metabolites and control gene expression by modifying their structure. As riboswitches often control essential genes in pathogenic bacteria, riboswitches have been proposed as new targets for antibiotics. High-throughput screening provides a powerful approach to identify riboswitch ligand analogs that could act as powerful antibacterial drugs. Biochemical assays have already been used to find riboswitch-binding analogs, but those methods do take into account the transcriptional context for riboswitch regulation. As the importance of co-transcriptional ligand binding has been shown for several riboswitches, it is vital to develop an assay that screens riboswitch-binding analogs during the transcriptional process. Here, we describe the development of a dual molecular beacon system monitoring the transcriptional regulation activity of the Bacillus subtilis pbuE adenine riboswitch. This system relies on two molecular beacons that enable the monitoring of transcription efficiency, as well as the regulatory activity of the riboswitch. Different analogs were tested using our system, and a good correlation was observed between riboswitch activity and reported metabolite affinities. This method is specific, reliable and could be applied at the high-throughput level for the identification of new potential antibiotics targeting any riboswitch-regulating gene expression at the mRNA level.