Nucleic Acid-Templated Synthesis of Cationic Styryl Dyes in Vitro and in Living Cells.

A novel method for synthesizing cationic styryl dyes through a nucleic acid-templated reaction has been developed. This approach overcomes issues associated with traditional synthesis methods, such as harsh conditions, low throughput, and wasteful chemicals. The presence of a nucleic acid template accelerated the styryl dye formation from quaternized heteroaromatic and cationic aldehyde substrates. These styryl dyes show remarkable optical properties change when bound to nucleic acids, hence the success of the synthesis could be readily monitored in situ by UV-Vis and fluorescence spectroscopy and the optical properties data were also observable at the same time. This method provides the desired products from a broad range of coupling partners. By employing different substrates and templates, it is possible to identify new dyes that can bind to a specific type of nucleic acid such as a G-quadruplex. The templated dye synthesis is also successfully demonstrated in live HeLa cells. This approach is a powerful tool for the rapid synthesis and screening of dyes specific for diverse types of nucleic acids or cellular organelles, facilitating new biological discoveries.

A quest for novel antimicrobial targets: Inhibition of Asp-tRNAAsn/Glu-tRNAGln amidotransferase (GatCAB) by synthetic analogs of aminoacyl-adenosine in vitro and live bacteria.

The Asp-tRNAAsn/Glu-tRNAGln amidotransferase (GatCAB) has been proposed as a novel antibacterial drug target due to its indispensability in prominent human pathogens. While several inhibitors with in vitro activity have been identified, none have been demonstrated to have potent activity against live bacteria. In this work, seven non-hydrolyzable transition state mimics of GatCAB were synthesized and tested as the transamidase inhibitors against GatCAB from the human pathogen Helicobacter pylori. Notably, the methyl sulfone analog of glutamyl-adenosine significantly reduced GatCAB's transamination rate. Additionally, four lipid-conjugates of these mimics displayed antibacterial activity against Bacillus subtilis, likely due to enhanced cell permeability. Inhibitory activity against GatCAB in live bacteria was confirmed using a sensitive gain-of-function dual luciferase reporter in Mycobacterium bovis-BCG. Only the lipid-conjugated methyl sulfone analog exhibited a significant increase in mistranslation rate, highlighting its cell permeability and inhibitory potential. This study provides insights for developing urgently needed novel antibacterial agents amidst emerging antimicrobial drug resistance.

Sequential Flow Controllable Microfluidic Device for G-Quadruplex DNAzyme-Based Electrochemical Detection of SARS-CoV-2 Using a Pyrrolidinyl Peptide Nucleic Acid.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a significant health issue globally. Point-of-care (POC) testing that can offer a rapid and accurate diagnosis of SARS-CoV-2 at the early stage of infection is highly desirable to constrain this outbreak, especially in resource-limited settings. Herein, we present a G-quadruplex DNAzyme-based electrochemical assay that is integrated with a sequential flow controllable microfluidic device for the detection of SARS-CoV-2 cDNA. According to the detection principle, a pyrrolidinyl peptide nucleic acid probe is immobilized on a screen-printed graphene electrode for capturing SARS-CoV-2 DNA. The captured DNA subsequently hybridizes with another DNA probe that carries a G-quadruplex DNAzyme as the signaling unit. The G-quadruplex DNAzyme catalyzes the H2O2-mediated oxidation of hydroquinone to benzoquinone that can be detected using square-wave voltammetry to give a signal that corresponds to the target DNA concentration. The assay exhibited high selectivity for SARS-CoV-2 DNA and showed a good experimental detection limit at 30 pM. To enable automation, the DNAzyme-based assay was combined with a capillary-driven microfluidic device featuring a burst valve technology to allow sequential sample and reagent delivery as well as the DNA target hybridization and enzymatic reaction to be operated in a precisely controlled fashion. The developed microfluidic device was successfully applied for the detection of SARS-CoV-2 from nasopharyngeal swab samples. The results were in good agreement with the standard RT-PCR method and could be performed within 20 min. Thus, this platform offers desirable characteristics that make it an alternative POC tool for COVID-19 diagnosis.

Focusing ion funnel-assisted ambient electrospray enables high-density and uniform deposition of non-spherical gold nanoparticles for highly sensitive surface-enhanced Raman scattering.

Surface-enhanced Raman scattering (SERS) is a powerful technique for detecting trace amounts of analytes. However, the performance of SERS substrates depends on many variables including the enhancement factor, morphology, consistency, and interaction with target analytes. In this study, we investigated, for the first time, the use of electrospray deposition (ESD) combined with a novel ambient focusing DC ion funnel to deposit a high density of gold nanoparticles (AuNPs) to generate large-area, uniform substrates for highly sensitive SERS analysis. We found that the combination of ambient ion focusing with ESD facilitated high-density and intact deposition of non-spherical NPs. This also allowed us to take advantage of a polydisperse colloidal solution of AuNPs (consisting of nanospheres and nanorods), as confirmed by finite-difference time domain (FDTD) simulations. Our SERS substrate exhibited excellent capture capacity for model analyte molecules, namely 4-aminothiophenol (4-ATP) and Rhodamine 6G (R6G), with detection limits in the region of 10-11 M and a relative standard deviation of <6% over a large area (∼500 × 500 µm2). Additionally, we assessed the quantitative performance of our SERS substrate using the R6G probe molecule. The results demonstrated excellent linearity (R2 > 0.99) over a wide concentration range (10-4 M to 10-10 M) with a detection limit of 80 pM.

Cyanostilbene-based fluorescent paper array for monitoring fish and meat freshness via amino content detection.

The detection of biogenic amines released from degraded meats is an effective method for evaluating meat freshness. However, existing traditional methods like titration are deemed tedious, while the use of sophisticated analytical instruments is not amenable to field testing. Herein, a cyanostilbene-based fluorescent array was rapidly fabricated using macroarray synthesis on a cellulose paper surface to detect amines liberated from spoiled beef, fish, and chicken. The fluorescence changes of immobilized molecules from the interaction with gaseous amines were used to monitor changes in freshness levels. Thanks to the high-throughput nature of macroarray synthesis, a set of highly responsive molecules such as pyridinium and dicyanovinyl moieties were quickly revealed. Importantly, this method offers flexibility in sensing applications including (1) sensing by individual sensor molecules, where the fluorescence response correlated well with established titration methods, and (2) collective sensing whereby chemometric analysis was used to provide a cutoff of freshness with 73-100% accuracy depending on meat types. Overall, this study paves the way for a robust and cost-effective tool for monitoring meat freshness.

Surfactant-Assisted Ozonolysis of Alkenes in Water: Mitigation of Frothing Using Coolade as a Low-Foaming Surfactant.

Aqueous-phase ozonolysis in the atmosphere is an important process during cloud and fog formation. Water in the atmosphere acts as both a reaction medium and a reductant during the ozonolysis. Inspired by the atmospheric aqueous-phase ozonolysis, we herein report the ozonolysis of alkenes in water assisted by surfactants. Several types of surfactants, including anionic, cationic, and nonionic surfactants, were investigated. Although most surfactants enhanced the solubility of alkenes in water, they also generated excessive foaming during the ozone bubbling, which led to the loss of products. Mitigation of the frothing was accomplished by using Coolade as a nonionic and low-foaming surfactant. Coolade-assisted ozonolysis of alkenes in water provided the desired carbonyl products in good yields and comparable to those achieved in organic solvents. During the ozonolysis reaction, water molecules trapped within the polyethylene glycol region of Coolade were proposed to intercept the Criegee intermediate to provide a hydroxy hydroperoxide intermediate. Decomposition of the hydroxy hydroperoxide led to formation of the carbonyl product without the need for a reductant typically required for the conventional ozonolysis using organic solvents. This study presents Coolade as an effective surfactant to improve the solubility of alkenes while mitigating frothing during the ozonolysis in water.

Assay Development and Identification of the First Plasmodium falciparum 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase Inhibitors.

In the fight towards eradication of malaria, identifying compounds active against new drug targets constitutes a key approach. Plasmodium falciparum 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase (PfHPPK) has been advanced as a promising target, as being part of the parasite essential folate biosynthesis pathway while having no orthologue in the human genome. However, no drug discovery efforts have been reported on this enzyme. In this study, we conducted a three-step screening of our in-house antifolate library against PfHPPK using a newly designed PfHPPK-GFP protein construct. Combining virtual screening, differential scanning fluorimetry and enzymatic assay, we identified 14 compounds active against PfHPPK. Compounds' binding modes were investigated by molecular docking, suggesting competitive binding with the HMDP substrate. Cytotoxicity and in vitro ADME properties of hit compounds were also assessed, showing good metabolic stability and low toxicity. The most active compounds displayed low micromolar IC50 against drug-resistant parasites. The reported hit compounds constitute a good starting point for inhibitor development against PfHPPK, as an alternative approach to tackle the malaria parasite.

Amplification-free DNA Sensor for the One-Step Detection of the Hepatitis B Virus Using an Automated Paper-Based Lateral Flow Electrochemical Device.

Until now, an electrochemical lateral flow assay (eLFA) capable of detecting nucleic acids has remained a challenge and has been scarcely explored because of its complicated multistep nature. Here, we report an automated paper-based eLFA device for the quantitative detection of the hepatitis B virus (HBV)-the major cause of liver cirrhosis and hepatocellular carcinoma (HCC). Using a time-delayed microfluidic strategy fabricated on paper, an automated and precisely sequenced solution transfer was enabled by single sample loading. A gold metallization strategy was employed for the signal-on electrochemical detection of the target DNA. Furthermore, a pyrrolidinyl peptide nucleic acid (so-called "acpcPNA") was used as a probe in this study because it offers higher specificity and yields lower background currents than those of traditional probes. Under optimal conditions, a broad dynamic range (10 pM to 2 µM) with an excellent detection limit (down to 7.23 pM) was achieved. The overall operation can be completed within 7 min of sample loading. The proposed sensor was successfully applied in HBV DNA detection in sera from patients without any amplification step (e.g., PCR) required, thus simplifying the operation further. Additionally, the results obtained from this present device are in accordance with the standard real-time PCR, thus supporting the accuracy of the method.

Pyrrolidinyl Peptide Nucleic Acid Probes Capable of Crosslinking with DNA: Effects of Terminal and Internal Modifications on Crosslink Efficiency.

In this study, we describe a furan-modified acpcPNA as a probe that can form an interstrand crosslink (ICL) with its DNA target upon activation with N-bromosuccinimide (NBS). To overcome the problem of furan instability under acidic conditions, a simple and versatile post-synthetic methodology for the attachment of the furan group to the PNA probe was developed. Unlike in other designs, the furan was placed at the end of the PNA molecule or tethered to the PNA backbone with all the base pairs in the PNA ⋅ DNA duplexes fully preserved. Hence, the true reactivity of each nucleobase towards the crosslinking could be compared. We show that all DNA bases except T could participate in the crosslinking reaction when the furan was placed at the end of the PNA strand. The crosslinking process was sensitive to mispairing, and lower crosslinking efficiency was observed in the presence of a base-mismatch in the PNA ⋅ DNA duplex. In contrast, when the furan was placed at internal positions of the acpcPNA ⋅ DNA duplex, no ICL was observed; this was explained by the inability of a hydrogen-bonded nucleobase to participate in the crosslinking reaction. The crosslinking efficiency was considerably improved, despite lower duplex stability, when an unpaired base (in the form of C-insertion) was present in the complementary DNA strand close to the furan modification site.

Isothermal Detection of Canine Blood Parasite (Ehrlichia canis) Utilizing Recombinase Polymerase Amplification Coupled with Graphene Oxide Quenching-Based Pyrrolidinyl Peptide Nucleic Acid.

Canine monocytic ehrlichiosis (CME), caused by transmitted Ehrlichia canis infection, is a major disease in dogs with worldwide distribution. Herein, a nucleic acid assay was established for the identification of E. canis infection employing a fluorescently labeled conformationally constrained pyrrolidinyl PNA probe (Flu-acpcPNA) designed to sequence-specifically target the 16S rRNA gene. The sensing principle is based on the excellent quenching ability of graphene oxide (GO) of the free PNA probe, that was diminished upon binding to the DNA target. The addition of DNase I improved the performance of the detection system by eliminating the nonspecific quenching capability of long-chain dsDNA and thus enhancing the fluorescence signaling. The assay was coupled with a recombinase polymerase amplification (RPA) technique, which could be performed under isothermal conditions (37 °C) without DNA denaturation and purification steps. The established method is simple to set up and execute, proving a rapid, specific, and sensitive detection of 16S rRNA gene of E. canis with a limit of detection at least 11.1 pM. This technique shows good potential for the visual detection of double-stranded DNA targets without the need for PCR or complicated instruments, which shows great promise for practical usage in resource limited areas.

Pyrrolidinyl peptide nucleic acids bearing hydroxy-modified cyclobutane building blocks: Synthesis and binding properties.

The conformationally constrained pyrrolidinyl PNA with a dipeptide consisting of an alternating nucleobase-modified D-proline and a cyclic ß-amino acid "spacer" exhibited improved nucleic acid binding properties compared to the original PNA. The pyrrolidinyl PNA with the four-membered ring spacer (1S,2S)-2-aminocyclobutanecarboxylic acid (acbcPNA) are among the best performed members of the pyrrolidinyl PNA family. However, these PNA suffer some limitations such as aqueous solubility and non-specific interactions due to their extreme hydrophobicity. In the present work, a hydroxy group is introduced onto the cyclobutane ring spacer of the acbcPNA with the aim of decreasing its hydrophobicity. To this end, a Fmoc/tBu ether-protected 4-hydroxy-2-aminocyclobutanecarboxylic acid building block was synthesized and resolved by chiral HPLC. Each enantiomer was used to synthesize the hydroxy-modified acbcPNA employing Fmoc solid-phase peptide synthesis. DNA/RNA binding studies indicated that the introduction of the hydroxy group to the acbcPNA decreases the binding affinity toward complementary DNA and RNA while maintaining the sequence and directional specificity of unmodified acbcPNA. The hydrophobicity of the hydroxy-modified acbcPNA decreased with the number of hydroxy groups added as indicated by the decrease in the logP values. Only two modifications were sufficient to decrease the logP by an order of magnitude without excessively lowering the binding affinity nor the specificity. This work thus demonstrated that the specific structural modifications for this type of PNA model can be performed in a modular fashion, which paves the way toward the future realization of improving hydrophilicity and nucleic acid binding affinity as well as specificity.

Surfactant-mediated thioglycosylation of 1-hydroxy sugars in water.

Thioglycosides are an important class of sugars, since they can be used as non-ionic biosurfactants, biomimetic glycosides, and building blocks for carbohydrate synthesis. Previously, Brønsted- or Lewis-acid-catalyzed dehydrative glycosylations between a 1-hydroxy sugar and a thiol have been reported to yield open-chain dithioacetal sugars as the major products instead of the desired thioglycosides. These dithioacetal sugars are by-products derived from the endocyclic bond cleavage of the thioglycosides. Herein, we report dehydrative glycosylation in water mediated by a Brønsted acid-surfactant combined catalyst (BASC). Glycosylations between 1-hydroxy furanosyl/pyranosyl sugars and primary, secondary, and tertiary aliphatic/aromatic thiols in the presence of dodecyl benzenesulfonic acid (DBSA) provided the thioglycoside products in moderate to good yields. Microwave irradiation led to improvements in the yields and a shortening of the reaction time. Remarkably, open-chain dithioacetal sugars were not detected in the DBSA-mediated glycosylations in water. This method is a simple, convenient, and rapid approach to produce a library of thioglycosides without the requirement of anhydrous conditions. Moreover, this work also provides an excellent example of complementary reactivity profiles of glycosylation in organic solvents and water.

Paper-based sensor from pyrrolidinyl peptide nucleic acid for the efficient detection of Bacillus cereus.

Bacillus cereus is one of the most common foodborne pathogens found in various kinds of staple foods such as rice and wheat. A rapid and accurate detection method for this pathogen is highly desirable for the sustainable production of relevant food products. While several classical and molecular-based detection methods are available for the identification of B. cereus, they suffered one or more limitations such as the requirement for a tedious and time-consuming process, less than ideal specificity, and the lack of portability. Herein, we developed the first paper-based sensing device that exhibits high species specificity with sufficiently low limit of detection for the visual detection of specific DNA sequences of B. cereus. The success is attributed to the strategic planning of fabrication in various dimensions including thorough bioinformatics search for highly specific genes, the use of the pyrrolidinyl peptide nucleic acid (PNA) probe whose selectivity advantage is well documented, and an effective PNA immobilization and DNA-binding visualization method with an internal cross-checking system for validating the results. Testing in rice matrices indicates that the sensor is capable of detecting and distinguishing B. cereus from other bacterial species. Hence, this paper-based sensor has potential to be adopted as a practical means to detect B. cereus in food industries.

Flexible Drift Tube for High Resolution Ion Mobility Spectrometry (Flex-DT-IMS).

This paper describes, in detail, the development of a novel, low-cost, and flexible drift tube (DT) along with an associated ion mobility spectrometer system. The DT is constructed from a flexible printed circuit board (PCB), with a bespoke "dog-leg" track design, that can be rolled up for ease of assembly. This approach incorporates a shielding layer, as part of the flexible PCB design, and represents the minimum dimensional footprint conceivable for a DT. The low thermal mass of the polyimide substrate and overlapping electrodes, as afforded by the dog-leg design, allow for efficient heat management and high field linearity within the tube-achieved from a single PCB. This is further enhanced by a novel double-glazing configuration which provides a simple and effective means for gas management, minimizing thermal variation within the assembly. Herein, we provide a full experimental characterization of the flexible DT ion mobility spectrometer (Flex-DT-IMS) with corresponding electrodynamic (Simion 8.1) and fluid dynamic (SolidWorks) simulations. The Flex-DT-IMS is shown to have a resolution >80 and a detection limit of low nanograms for the analysis of common explosives (RDX, PETN, HMX, and TNT).

Modulation of DNA and RNA by PNA.

Peptide nucleic acid (PNA), a synthetic DNA mimic that is devoid of the (deoxy)ribose-phosphate backbone yet still perfectly retains the ability to recognize natural nucleic acids in a sequence-specific fashion, can be employed as a tool to modulate gene expressions via several different mechanisms. The unique strength of PNA compared to other oligonucleotide analogs is its ability to bind to nucleic acid targets with secondary structures such as double-stranded and quadruplex DNA as well as RNA. This digest aims to introduce general readers to the advancement in the area of modulation of DNA/RNA functions by PNA, its current status and future research opportunities, with emphasis on recent progress in new targeting modes of structured DNA/RNA by PNA and PNA-mediated gene editing.

Quenching of fluorescently labeled pyrrolidinyl peptide nucleic acid by oligodeoxyguanosine and its application in DNA sensing.

Quenching by nucleobases can significantly affect the fluorescence properties of many fluorophores. The quenching efficiency depends on the electronic properties of the fluorophore and adjacent nucleobases. In this study, we present a hitherto unreported high-efficiency quenching (up to 90%) of various fluorescently labeled pyrrolidinyl peptide nucleic acid (acpcPNA) probes by oligodeoxyguanosine (dGX). The quenching principle relies on the electrostatic interaction between the positively charged lysine-modified acpcPNA probe and the negatively charged oligodeoxyguanosine. The addition of stoichiometric quantities of a DNA target with the sequence complementary to the PNA probe restored the fluorescence to the original level. This was explained by the binding of the DNA to the PNA via a specific base pairing, which resulted in the separation of the oligodeoxyguanosine quencher from the fluorophore. Much less fluorescence restoration was observed in the DNA containing one or more mismatched bases. Applications of the oligodeoxyguanosine-quenched PNA probes for DNA sequence determination, including in multiplex formats, are demonstrated. The performance in terms of sensitivity and mismatch discrimination is comparable to classical PNA-based molecular beacons but without the need for double-labeling, which is expensive and presents solubility issues, or a dedicated quencher probe. This exemplifies a novel use of the unique electrostatic properties of PNA to develop a DNA sensing platform for DNA sequence determination.

Synthesis and antileishmanial activity of fluorinated rhodacyanine analogues: The 'fluorine-walk' analysis.

In a search for potent antileishmanial drug candidates, eighteen rhodacyanine analogues bearing fluorine or perfluoroalkyl substituents at various positions were synthesized. These compounds were tested for their inhibitory activities against Leishmania martiniquensis and L. orientalis. This 'fluorine-walk' analysis revealed that the introduction of fluorine atom at C-5, 6, 5', or 6' on the benzothiazole units led to significant enhancement of the activity, correlating with the less negative reduction potentials of the fluorinated analogues confirmed by the electrochemical study. On the other hand, CF3 and OCF3 groups were found to have detrimental effects, which agreed with the poor aqueous solubility predicted by the in silico ADMET analysis. In addition, some of the analogues including the difluorinated species showed exceptional potency against the promastigote and axenic amastigote stages (IC50 = 40-85 nM), with the activities surpassing both amphotericin B and miltefosine.

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.

Clickable styryl dyes for fluorescence labeling of pyrrolidinyl PNA probes for the detection of base mutations in DNA.

Fluorescent hybridization probes are important tools for rapid, specific and sensitive analysis of genetic mutations. In this work, we synthesized novel alkyne-modified styryl dyes for conjugation with pyrrolidinyl peptide nucleic acid (acpcPNA) by click chemistry for the development of hybridization responsive fluorescent PNA probes. The free styryl dyes generally exhibited weak fluorescence in aqueous media, and the fluorescence was significantly enhanced (up to 125-fold) upon binding with DNA duplexes. Selected styryl dyes that showed good responses with DNA were conjugated with PNA via sequential reductive alkylation-click chemistry. Although these probes showed little fluorescence change when hybridized to complementary DNA, significant fluorescence enhancements were observed in the presence of structural defects including mismatched, abasic and base-inserted DNA targets. The largest increase in fluorescence quantum yield (up to 14.5-fold) was achieved with DNA carrying base insertion. Although a number of probes were designed to give fluorescence response to complementary DNA targets, probes that are responsive to mutations such as single nucleotide polymorphism (SNP), base insertion/deletion and abasic site are less common. Therefore, styryl-dye-labeled acpcPNA is a unique probe that is responsive to structural defects in the duplexes that may be further applied for diagnostic purposes.

Facile conversion of ATP-binding RNA aptamer to quencher-free molecular aptamer beacon.

We have developed RNA-based quencher-free molecular aptamer beacons (RNA-based QF-MABs) for the detection of ATP, taking advantage of the conformational changes associated with ATP binding to the ATP-binding RNA aptamer. The RNA aptamer, with its well-defined structure, was readily converted to the fluorescence sensors by incorporating a fluorophore into the loop region of the hairpin structure. These RNA-based QF-MABs exhibited fluorescence signals in the presence of ATP relative to their low background signals in the absence of ATP. The fluorescence emission intensity increased upon formation of a RNA-based QF-MAB·ATP complex.