Recent advances in the development of graphene-based surface plasmon resonance (SPR) interfaces

Surface plasmon resonance (SPR) is a powerful technique for measurement of biomolecular interactions in real-time in a label-free environment. One of the most common techniques for plasmon excitation is the Kretschmann configuration, and numerous studies of ligand–analyte interactions have been performed on surfaces functionalized with a variety of biomolecules, for example DNA, RNA, glycans, proteins, and peptides. A significant limitation of SPR is that the substrate must be a thin metal film. Post-coating of the metal thin film with a thin dielectric top layer has been reported to enhance the performance of the SPR sensor, but is highly dependent on the thickness of the upper layer and its dielectric constant. Graphene is a single-atom thin planar sheet of sp2 carbon atoms perfectly arranged in a honeycomb lattice. Graphene and graphene oxide are good supports for biomolecules because of their large surface area and rich π conjugation structure, making them suitable dielectric top layers for SPR sensing. In this paper, we review some of the key issues in the development of graphene-based SPR chips. The actual challenges of using these interfaces for studying biomolecular interactions will be discussed and the first examples of the use of graphene-on-metal SPR interfaces for biological sensing will be presented.     

Electrophoretic characterization of LNP/AAV-encapsulated nucleic acids: Strengths and weaknesses

The use of nucleic acids (NAs) has revolutionized medical approaches and ushered in a new era of combating various diseases. Accordingly, there is an increasing demand for accurate identification, localization, quantification, and characterization of NAs encapsulated in nonviral or viral vectors. The vast spectrum of molecular dimensions and intra- and intermolecular interactions presents a formidable obstacle for NA analytical development. Typically, the comprehensive analysis of encapsulated NAs, free NAs, and their spatial distribution poses a challenge that is seldom tackled in its complete complexity. The identification of appropriate physicochemical methodologies for large nonencapsulated or encapsulated NAs is particularly intricate and necessitates an evaluation of the analytical outcomes and their appropriateness in addressing critical quality attributes. In this work, we examine the analytics of non-encapsulated or encapsulated large NAs (>500 nucleotides) utilizing capillary electrophoresis (CE) and liquid chromatography (LC) methodologies such as free zone CE, gel CE, affinity CE, and ion pair high-performance liquid chromatography (HPLC). These methodologies create a complete picture of the NA's critical quality attributes, including quantity, identity, purity, and content ratio.     

Nitroanilines as Quenchers of Pyrene Fluorescence

The quenching of pyrene and 1‐methylpyrene fluorescence by nitroanilines (NAs), such as 2‐, 3‐, and 4‐nitroaniline (2‐NA, 3‐NA, and 4‐NA, respectively), 4‐methyl‐3‐nitroaniline (4‐M ‐3‐NA), 2‐methyl‐4‐nitroaniline (2‐M‐4‐NA), and 4‐methyl‐3,5‐dinitroaniline (4‐M‐3,5‐DNA), are studied in toluene and 1,4‐dioxane. Steady‐state fluorescence data show the higher efficiency of the 4‐NAs as quenchers and fit with a sphere‐of‐action model. This suggests a 4‐NA tendency of being in close proximity to the fluorophore, which could be connected with their high polarity/hyperpolarizability. In addition, emission and excitation spectra evidence the formation of emissive pyrene—NA ground‐state complexes in the case of the 4‐NAs and, in a minor degree, in the 2‐NA. Moreover, time‐resolved fluorescence experiments show that increasing amounts of NA decrease the pyrene fluorescence lifetime to a degree that depends on the NA nature and is larger in the less viscous solvent (toluene). Although the NA absorption and the pyrene (Py) emission overlap, we found no evidence of dipole–dipole energy transfer from the pyrene singlet excited state (¹Py) to the NAs; this could be due to the low NA concentration used in these experiments. Transient absorption spectra show that the formation of the pyrene triplet excited state (³Py) is barely affected by the presence of the NAs in spite of their efficiency in ¹Py quenching, suggesting the involvement of ¹Py—NA exciplexes which—after intersystem crossing—decay efficiently into ³Py.     

An optical surface plasmon resonance biosensor for determination of tetanus toxin

Surface plasmon resonance (SPR) has been successfully applied for the simple, rapid, and label-free assay of various biomolecules. This assay evaluates a novel wavelength modulation SPR biosensor for the detection of tetanus toxin. The wavelength modulation SPR biosensor is designed based on fixing the incident angle of light and measuring the reflected intensities in the resonance wavelength range spanning 400-800 nm simultaneously. Tetanus toxin (TeNT), one of the most potent toxins known, is synthesized as a 150 kDa single polypeptide chain. The SPR biosensor has been shown to be capable of directly detecting concentration of tetanus toxin as low as 0.028 Lf ml⁻¹. Under selected experimental conditions, the SPR biosensor has a good reproducibility, sensitivity and reversibility. The results illustrate how wavelength modulation SPR biosensor can be used to detect biomolecular interactions.     

Microfluidic Isolation of Nucleic Acids

The detection of nucleic acids (NAs) within micro total analysis systems (μTASs) for point‐of‐care use is a rapidly developing research area. The efficient isolation of NAs from a raw sample is crucial for these systems to be maximally effective. The use of microfluidics assists in reducing sample sizes and reagent consumption, increases speed, avoids contamination, and enables automation. Through miniaturization into microchips, new techniques have been realized that would be unfavorable and inconvenient to use on a macroscopic scale, but provide an excellent platform for the purification of NAs on a microscopic scale. This Review considers the complexities of NA isolation with miniaturized and microfluidic devices, as well as the considerations when choosing a technique for microfluidic NA isolation, along with their advantages, disadvantages, and potential applications. The techniques presented include using silica‐based surfaces, functionalized paramagnetic beads, oligonucleotide‐modified polymer surfaces, pH‐dependent charged surfaces, Al₂O₃ membranes, and liquid‐phase isolation. This Review provides a basis to develop the chemistry to improve NA isolation and move it toward achieving 100 % efficiencies.     

Carbon-on-metal films for surface plasmon resonance detection of DNA arrays

Surface plasmon resonance (SPR) imaging affords label-free monitoring of biomolecule interactions in an array format. A surface plasmon conducting metal thin film is required for SPR measurements. Gold thin films are traditionally used in SPR experiments as they are readily functionalized with thiol-containing molecules through formation of a gold-sulfur bond. The lability of this gold-thiol linkage upon exposure to oxidizing conditions and ultraviolet light renders these surfaces incompatible with light-directed synthetic methods for fabricating DNA arrays. It is shown here that applying a thin carbon overlayer to the gold surface yields a chemically robust substrate that permits light-directed synthesis and also supports surface plasmons. DNA arrays fabricated on these carbon-metal substrates are used to analyze two classes of biomolecular interactions: DNA-DNA and DNA-protein. This new strategy allows the combinatorial study of binding interactions directly from native, unmodified biomolecules of interest and offers the possibility of discovering new ligands in complex mixtures such as cell lysates.     

Characterization and pattern recognition of oil-sand naphthenic acids using comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry

Oil-sand naphthenic acids (NAs) are organic wastes produced during the oil-sand digestion and extraction processes and are very difficult to separate and analyze as individual components due to their complex compositions. A comprehensive two-dimensional gas chromatography/time of flight mass spectrometry (GC x GC/TOF-MS) system was applied for the characterization of two commercial mixtures of naphthenic acids (Fluka and Acros) and a naphthenic acid sample extracted from the Syncrude tailings. Contour plots of chromatographic distributions of different Z homologous series of the Fluka, Acros and Syncrude NAs were constructed using fragment ions that were characteristic of the NA's molecular structures. Well-ordered patterns were observed for NAs of Z= 0 and -2 which corresponded to acyclic acids and monocyclic acids, respectively. For NAs of Z= -4, -6, and -8, specific zones were observed which would allow the pattern recognition of these NAs obtained from different origins. As expected, gas chromatographic retention times increase with the number of the carbons and the number of rings in the molecules. Little signal was obtained for NAs with Z numbers of -10, or lower. Deconvoluted mass spectra of various NA isomers were derived from the reconstructed GC x GC chromatogram, permitting detailed structural elucidations for NAs in the future. The current study demonstrated that the combination of GC x GC and the TOF-MS is a powerful to identify origins of the NAs in an effective manner. GC x GC/TOF-MS alone, however, may not be enough to characterize each individual isomer in a complex mixture such as NAs. The use of mass deconvolution software followed by library search have thus become necessary to separate and study the mass spectrum of each individual NA component, allowing a detailed identification of the toxic components within the NAs mixture.     

Determination of dissolved naphthenic acids in natural waters by using negative-ion electrospray mass spectrometry

Naphthenic acids (NAs) have been implicated as some of the most toxic substances in oil sands leachates and identified as priority substances impacting on aquatic environments. As a group of compounds, NAs are not well characterized and comprise a large group of saturated aliphatic and alicyclic carboxylic acids found in hydrocarbon deposits (petroleum, oil sands bitumen, and crude oils). Described is an analytical method using negative-ion electrospray ionization mass spectrometry (ES/MS) of extracts. Preconcentration was achieved by using a solid-phase extraction procedure utilizing a crosslinked polystyrene-based polymer with acetonitrile elution. Recovery of the Fluka Chemicals NA mixture was highly pH-dependent, with 100% recovery at pH 3.0, but only 66 and 51% recoveries at pHs 7 and 9, respectively. The dissolved phase of the NA was very dependent on sample pH. It is thus critical to measure the pH and determine the appropriate mass profiles to identify NAs in natural waters. The ES/MS analytical procedure proved to be a fast and sensitive method for the recovery and detection of NAs in natural waters, with a detection limit of 0.01 mg/L.     

Surface plasmon resonance sensors for real-time detection of cyclic citrullinated peptide antibodies

Surface plasmon resonance (SPR) sensors have been used for detection of various biomolecules because of their simplicity, high specificity and sensitivity, real-time detection, low cost, and no requirement of labeling. Recently, molecularly imprinted polymers that are easy to prepare, less expensive, stable, have talent for molecular recognition and also are used for creation selective binding sites for target molecule on the SPR sensors. Here, we show that preparation of cyclic citrullinated peptide antibody (anti-CCP) imprinted SPR sensor to detect CCP antibodies. For this purpose, anti-CCP/AAm pre-complex was synthesized by interacting acrylamide (AAm) monomer with anti-CCP. Then, anti-CCP imprinted (anti-CCP/PAAm) SPR sensor was obtained by reacting with anti-CCP/AAm pre-complex in the presence of the crosslinker, and initiator/activator pair. Besides this, non-imprinted (PAAm) SPR sensor was also prepared without using anti-CCP template. The SPR sensors were characterized and then adsorption-desorption studies were performed with pH 7.0 phosphate buffer (10 mM) and acetic acid (10%) with Tween 20 (1%) in pH 7.0 phosphate buffer. Selectivitiy of sensors was investigated by using immunoglobulin M (IgM) and bovine serum albumin (BSA). To determine the adsorption model of interactions between anti-CCP solutions and anti-CCP/PAAm SPR sensor, different adsorption models were performed. The calculated maximum reflection, detection limit, association and dissociation constants were 1.079 RU/mL, 0.177 RU/mL, 0.589 RU/mL and 1.697 mL/RU, respectively. Repeatability experiments of anti-CCP/PAAm SPR sensor was performed four times with adsorption-desorption-regeneration cycles without any performance losing. Results showed that anti-CCP/PAAm SPR sensor had high selectivity and sensitivity for detection of CCP antibodies.     

Recent advances in the preparation of glycopolymer bioconjugates

The significant progress made in understanding the role of carbohydrates and carbohydrates based therapeutics at molecular level has highlighted the importance of carbohydrate bioconjugates in the field of biology, chemistry and therapeutics. The glycosylation of biomolecules is a nature-inspired approach, to impart structural and functional properties to the biomolecules. The availability of facile techniques to synthesize well-defined glycopolymers of varying molecular weights, compositions and shape and their facile conjugation with biomolecules of interest have helped researchers in understanding many aspects of their biological functions at the molecular level. This review focuses on the development of glycopolymer-bioconjugates and provides a comprehensive overview of the present bioconjugation tools for their synthesis. The glycosylation of biomolecules is achieved by either pre or post-polymerization modification approaches. The review highlights the potential of living radical polymerization for the facile synthesis of glycopolymer bioconjugates using both pre and post-polymerization bioconjugation approaches, and without disrupting the native structure and functions of the biological molecules. Non-covalent carbohydrate–carbohydrate and carbohydrate–protein interactions play a significant role in many biological and pathological events. The non-covalent interactions of synthetic glycopolymers with biomolecules are also discussed in this review.     

Comparison of high- and low-resolution electrospray ionization mass spectrometry for the analysis of naphthenic acid mixtures in oil sands process water

The oil sands regions of Northern Alberta, Canada, contain an estimated 1.7 trillion barrels of oil in the form of bitumen, representing the second largest deposit of crude oil in the world. A rapidly expanding industry extracts surface-mined bitumen using alkaline hot water, resulting in large volumes of oil sands process water (OSPW) that must be contained on site due to toxicity. The toxicity has largely been attributed to naphthenic acids (NAs), a complex mixture of naturally occurring aliphatic and (poly-)alicyclic carboxylic acids. Research has increasingly focused on the environmental fate and remediation of OSPW NAs, but an understanding of these processes necessitates an analytical method that can accurately characterize and quantify NA mixtures. Here we report results of an interlaboratory comparison for the analysis of pure commercial NAs and environmental OSPW NAs using direct injection electrospray ionization mass spectrometry (ESI-MS) and high-pressure liquid chromatography/high-resolution mass spectrometry (HPLC/HRMS). Both methods provided very similar characterization of pure commercial NA mixture; however, the m/z selectivity of HPLC/HRMS was essential to prevent substantial false-positive detections and misclassifications in OSPW NA mixtures. For a range of concentrations encompassing those found in OSPW (10-100 mg/L), both methods produced linear response, although concentrations of commercial NAs above 50 mg/L resulted in slight non-linearity by HPLC/HRMS. A three-fold lower response factor for total OSPW NAs by HPLC/HRMS was largely attributable to other organic compounds in the OSPW, including hydroxylated NAs, which may explain the substantial misclassification by ESI-MS. For the quantitative analysis of unknown OSPW samples, both methods yielded total NA concentrations that correlated with results from Fourier transform infrared (FTIR), but the coefficients of determination were not high. Quantification by either MS method should therefore be considered semi-quantitative at best, albeit either method has substantial value in environmental fate experiments where relative concentration changes are the desired endpoints rather than absolute concentrations.     

Magnetic Fe3O4@Au composite-enhanced surface plasmon resonance for ultrasensitive detection of magnetic nanoparticle-enriched α-fetoprotein

Small molecules or analytes present at low concentrations are difficult to detect directly using conventional surface plasmon resonance (SPR) techniques because only small changes in the refractive index of the medium are typically induced by the binding of these analytes. Here, we present an amplification technique using core–shell Fe₃O₄@Au magnetic nanoparticles (MNPs) for an SPR bioassay. To evaluate this amplification effect, a novel SPR sensor based on a sandwich immunoassay was developed to detect α-fetoprotein (AFP) by immobilizing a primary AFP antibody (Ab₁) on the surface of a 3-mercapto-1-propanesulfonate/chitosan-ferrocene/Au NP (MPS/CS-Fc/Au NP) film employing Fe₃O₄@Au–AFP secondary antibody conjugates (Fe₃O₄@Au–Ab₂) as the amplification reagent. The stepwise fabrication of the biosensor was characterized using UV-vis spectroscopy, electrochemical impedance spectroscopy, and cyclic voltammetry. A calibration curve of Fe₃O₄@Au–Ab₂ conjugates amplification for AFP detection was obtained to yield a correlation in the range of 1.0–200.0 ng mL⁻¹ with a detection limit of 0.65 ng mL⁻¹, and a significant increase in sensitivity was therefore afforded through the use of Fe₃O₄@Au–Ab₂ conjugates as an amplifier. This magnetic separation and amplification strategy has great potential for the detection of other biomolecules of interest with low interference and high sensitivity by changing the antibody label used in the Fe₃O₄@Au–antibody conjugates.     

Molecular profiling of naphthenic acids in technical mixtures and oil sands process‐affected water using polar reversed‐phase liquid chromatography–mass spectrometry

In this work, a reversed‐phase ultra‐HPLC (UHPLC) ultrahigh resolution MS (UHRMS) method was evaluated for the comprehensive profiling of NAs containing two O atoms in each molecule (O2NAs; general formula C_nH_{2n + z}O₂, where n is the number of carbon atoms and z represents hydrogen deficiency). Using a polar cyanopropyl‐bonded phase column and negative‐ion electrospray ionization mass spectrometric detection at 120,000 FWHM (m/z 400), 187 and 226 O2NA species were found in two naphthenic acid technical mixtures, and 424 and 198 species with molecular formulas corresponding to O2NAs were found in two oil sands process‐affected water samples (one from a surface mining operation and the other from a steam‐assisted gravity drainage operation), respectively. To our knowledge, these are the highest numbers of molecular compositions of O2NAs that have been profiled thus far in environmental samples. Assignments were based on accurate mass measurements (≤3 ppm) combined with rational molecular formula generation, correlation of chromatographic behavior of O2NA homologues with their elemental compositions, and confirmation with carboxyl group‐specific chemical derivatization using 3‐nitrophenylhydrazine. Application of this UHPLC–UHRMS method to the quantitation of O2NAs in the surface mining operation‐derived water sample showed excellent linearity (R² = 0.9999) with external calibration, a linear range of 256‐fold in concentration, and quantitation accuracies of 64.9 and 69.4% at two “standard substance” spiking levels.     

A derivatisation and liquid chromatography/electrospray ionisation multistage mass spectrometry method for the characterisation of naphthenic acids

Naphthenic acids (NAs) are partially uncharacterised complex mixtures of carboxylic acids, resulting from the microbial oxidation of petroleum hydrocarbons. They are associated with the fouling of pipelines and process equipment in oil production and with corrosion in oil refineries. As by-products of the rapidly expanding oil (tar) sands industries, NAs are also pollutants and have proved to be toxic to a range of organisms. They also have important beneficial uses as fungicides, tyre additives and, paradoxically, also in the manufacture of corrosion inhibitors. These features make the characterisation of NAs an important goal for analytical chemists. Here we describe the synthesis of amide derivatives of NAs for characterisation by liquid chromatography/electrospray ionisation multistage mass spectrometry (LC/ESI-MS(n)). The method was applied to commercially available carboxylic acids, novel synthetic NAs, commercial NAs refined from crude oils, crude oil NAs and Athabasca oil sands NAs. In addition to confirming the number of alicyclic rings and length of alkyl side chain substituents (confirming information from existing methods), the MS(n) results provided further structural information. Most important of these was the finding that bi- to polycyclic acids containing ethanoate side chains, in addition to alkyl substituents, were widespread amongst the oil and oil sands NAs. The latter NAs are known end members of the beta-oxidation of NAs with even carbon number alkanoate chains. Since such NA mixtures are toxic, they should be targets for bioremediation. Bioremediation of NAs can also be monitored better by application of the methods described herein.     

NMF-Based Approach for Missing Values Imputation of Mass Spectrometry Metabolomics Data

In mass spectrometry (MS)-based metabolomics, missing values (NAs) may be due to different causes, including sample heterogeneity, ion suppression, spectral overlap, inappropriate data processing, and instrumental errors. Although a number of methodologies have been applied to handle NAs, NA imputation remains a challenging problem. Here, we propose a non-negative matrix factorization (NMF)-based method for NA imputation in MS-based metabolomics data, which makes use of both global and local information of the data. The proposed method was compared with three commonly used methods: k-nearest neighbors (kNN), random forest (RF), and outlier-robust (ORI) missing values imputation. These methods were evaluated from the perspectives of accuracy of imputation, retrieval of data structures, and rank of imputation superiority. The experimental results showed that the NMF-based method is well-adapted to various cases of data missingness and the presence of outliers in MS-based metabolic profiles. It outperformed kNN and ORI and showed results comparable with the RF method. Furthermore, the NMF method is more robust and less susceptible to outliers as compared with the RF method. The proposed NMF-based scheme may serve as an alternative NA imputation method which may facilitate biological interpretations of metabolomics data.     

Bundling mRNA Strands to Prepare Nano‐Assemblies with Enhanced Stability Towards RNase for In Vivo Delivery

Ribonuclease (RNase)‐mediated degradation of messenger RNA (mRNA) poses a huge obstruction to in vivo mRNA delivery. Herein, we propose a novel strategy to protect mRNA by structuring mRNA to prevent RNase attack through steric hinderance. Bundling of mRNA strands through hybridization of RNA oligonucleotide linkers allowed the preparation of mRNA nano‐assemblies (R‐NAs) comprised of 7.7 mRNA strands on average, mostly below 100 nm in diameter. R‐NA formation boosted RNase stability by around 100‐fold compared to naïve mRNA and preserved translational activity, allowing protein production. A mechanistic analysis suggests that an endogenous mRNA unwinding mechanism triggered by 5′‐cap‐dependent translation may induce selective R‐NA dissociation intracellularly, leading to smooth translation. R‐NAs showed efficient mRNA transfection in mouse brain, demonstrating the feasibility for in vivo administration.     

表面等离子体共振技术在蛋白质-蛋白质相互作用研究中的应用

表面等离子共振(SPR)近年来迅速发展为用于分析生物分子相互作用的一项技术.该技术无需标记、特异性强、灵敏度高、样品用量小,可实现在线连续实时检测.目前SPR已被广泛应用于免疫学、蛋白质组学、药物筛选、细胞信号转导、受体/配体垂钓等领域.该文阐述了基于表面等离子体共振技术生物传感器的基本原理和技术流程,综述了SPR在蛋白质-蛋白质相互作用动力学研究、蛋白质结构及功能研究、蛋白质突变和碎片分析、信号转导中的应用以及SPR在蛋白质-蛋白质相互作用研究中的多项关键技术.指出SPR通过与光谱、电化学等多技术联用后,可以获得更加详实的信息.     

SDS‐PAGE procedure: Application for characterization of new entirely uncharged nucleic acids analogs

SDS‐PAGE is considered to be a universal method for size‐based separation and analysis of proteins. In this study, we applied the principle of SDS‐PAGE to the analysis of new entirely uncharged nucleic acid (NA) analogues, – phosphoryl guanidine oligonucleotides (PGOs). The procedure was also shown to be suitable for morpholino oligonucleotides (PMOs) and peptide nucleic acids (PNAs). It was demonstrated that SDS can establish hydrophobic interactions with these types of synthetic NAs, giving them a net negative charge and thus making these molecules mobile in polyacrylamide slab gels under the influence of an electric field.     

Polymeric optical microscreen for high-resolution surface plasmon resonance microarray imaging

In this study, we demonstrate a simple method to fabricate surface plasmon resonance (SPR) imaging microarrays using polymer micropatterns. The use of a micrometer-scale polymeric optical screen (microPOS) passivates the region deposited with polymer by completely removing SPR signals or by saturating the SPR signal far beyond the detection range of SPR imaging. Two schemes were suggested to create a surface microPOS by either micropatterning a thick insulating layer before deposition of a metal layer (complete removal of SPR) or after deposition of a metal layer (saturation of SPR signal). The two schemes were successfully applied for the imaging of biological adsorption with a high imaging resolution of approximately 100 microm/pattern and 10 microm separation. The validity of the system was verified with a biotin-streptavidin system as a model for the systematic binding of biomolecules. Further, binding of prostate-specific antigen (PSA) onto the anti-PSA SPR microarray was demonstrated as a useful method for detecting a cancer marker.     

Naphthyridine‐Benzoazaquinolone: Evaluation of a Tricyclic System for the Binding to (CAG)n Repeat DNA and RNA

The expansion of CAG repeats in the human genome causes the neurological disorder Huntington's disease. The small‐molecule naphthyridine‐azaquinolone NA we reported earlier bound to the CAG/CAG motif in the hairpin structure of the CAG repeat DNA. In order to investigate and improve NA ‐binding to the CAG repeat DNA and RNA, we conducted systematic structure‐binding studies of NA to CAG repeats. Among the five new NA derivatives we synthesized, surface plasmon resonance (SPR) assay showed that all of the derivatives modified from amide linkages in NA to a carbamate linkage failed to bind to CAG repeat DNA and RNA. One derivative, NBzA , modified by incorporating an additional ring to the azaquinolone was found to bind to both d(CAG)₉ and r(CAG)₉. NBzA binding to d(CAG)₉ was similar to NA binding in terms of large changes in the SPR assay and circular dichroism (CD) as well as pairwise binding, as assessed by electron spray ionization time‐of‐flight (ESI‐TOF) mass spectrometry. For the binding to r(CAG)₉, both NA and NBzA showed stepwise binding in ESI‐TOF MS, and NBzA ‐binding to r(CAG)₉ induced more extensive conformational change than NA ‐binding. The tricyclic system in NBzA did not show significant effects on the binding, selectivity, and translation, but provides a large chemical space for further modification to gain higher affinity and selectivity. These studies revealed that the linker structure in NA and NBzA was suitable for the binding to CAG DNA and RNA, and that the tricyclic benzoazaquinolone did not interfere with the binding.