Label-free sensing platform for miRNA-146a based on chromo-fluorogenic pyrophosphate recognition

doi:10.1016/j.jinorgbio.2019.110867

Journal of Inorganic Biochemistry

Volume 203, February 2020, 110867

https://doi.org/10.1016/j.jinorgbio.2019.110867 Get rights and content

Highlights

•
Chromo-fluorescent Cu²⁺ probe, for miRNA-146a recognition based on PPi sensing platform
•
Dual mode of miRNA's recognition in physiological conditions
•
Robust and real time monitoring of miRNA amplification in modified biological buffers
•
nM and aM level sensitivity toward miRNA in colorimetric and fluorometric methods

Abstract

In this study we applied the dual-responsive chromo-fluorescent Cu²⁺ chelate 1C for the recognition of miRNA-146a through a pyrophosphate (PPi) sensing strategy in a rolling circle amplification (RCA) process. This approach for the recognition of miRNA-146a was highly robust, selective, and sensitive down to the attomolar (fluorogenic) and sub-micromolar (chromogenic) ranges under modified biochemical conditions at elevated temperature. Probe 1 selectively recognized Cu²⁺ and PPi ions in a sequential manner, as evidenced by colorless→pink→colorless transitions; the fluorescence emissions centered at 480 nm underwent a corresponding on–off–on sequence in the bluish-green region. We attribute this reversible switching upon the addition of Cu²⁺/PPi ions to effective chelation-induced ligand-to-metal charge/electron transfer that resulted in opening of the lactam ring upon complexation and closing of the lactam ring upon decomplexation. We also report a label-free approach for monitoring miRNA-146a amplification in an RCA process under modified T4 ligase and ϕ₂₉ buffer conditions, using the Cu²⁺ ensemble 1C at pH 7.0 (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid: HEPES, 10 mM MgCl₂); the time required to perform this process (40–50 min) was relatively shorter than conventional RCA process. This ensemble 1C could recognize miRNA-146a colorimetrically (from pink to colorless) and fluorimetrically (“turn-on” mode) at concentrations within the highly sensitive atto-/nanomolar range under physiological conditions. This cost-effective label-free sensing strategy appears to be a universal method for detecting miRNAs according to the specified length of the template.

Graphical abstract

Cu²⁺ ensembles as an invitro-diagnostic tool: Robust and novel chromo-fluorogenic method for identification of miRNAs (up to nM/μL and aM/μL range) in modified biological buffers based on pyrophosphate sensing platform.

Introduction

Label-free optical methods for the identification of genetic materials as vital targets have several advantages over labeling approaches-in particular, higher spatial and temporal resolutions relative to those obtainable using electro-analytical approaches, nuclear magnetic resonance (NMR) spectroscopy, high-performance liquid chromatography (HPLC), and mass spectrometry [1]. Labeling approaches require the synthesis of modified oligonucleotides complimentary to a particular analyte, with the function based on a Watson–Crick base-pairing mode (duplex) to cause structure-related photophysical changes. Although such processes can have high sensitivities and selectivities, the syntheses can be laborious and time-consuming, and expensive in terms of the chemicals consumed and the highly sophisticated instruments required for characterization [2,3]. Accordingly, in this study we have developed a label-free method to identify miRNAs-short non-coding single-stranded RNA sequences (18–24 nt) in the untranslated region of messenger RNA (mRNAs).

Generally, miRNAs are involved in gene regulation, through induced degradation of mRNA, thereby regulating protein translation. In addition, several miRNAs are also involved in cell proliferation, differentiation, apoptosis, hematopoiesis, and stress resistance, and also play roles in various diseases in humans and animals [3]. A recent investigation revealed that miRNAs are excellent biomarkers-they are stable in serum, plasma, and whole blood lines-and that they exist primarily in encapsulated form in the exosome [4]. Conventional strategies for miRNA sensing rely on a complimentary DNA/RNA (preferably DNA) sequence and fluorophore/quencher, fluorophore (quencher-free), or fluorophore/fluorophore (donor/acceptor pairs for fluorescence resonance energy transfer:FRET) units, in addition to signal amplification techniques [5]. In addition, several miRNA recognition approaches have been developed based on DNA–protein and RNA–protein interactions involving chromofluorescent labels [6]. These strategies typically provide outstanding selectivity toward a specific miRNA under ambient physiological conditions, even in complex biological fluids. In general, the use of labeled oligonucleotides/genetically modified fluorescent proteins requires highly aseptic media, buffers, and dissolved salts (cations or anions) to ensure modulation of the photophysical properties through weak noncovalent interactions-typically, conventional multiple hydrogen bonding (Hoogsteen or Watson–Crick mode), non-classical cation–π, anion–π, NH–π, and OH–π interactions, and van der Waals forces [7]. Exploiting such weak nonspecific interactions could increase the probability of false-positive signals when recognizing low concentrations of miRNA, piRNA (piwi interacting RNA), and mRNA (ca. 10⁻⁹–10⁻¹⁴ M) in complex biological fluids [8]. To gain high sensitivity and selectivity and allow qualitative analysis, signal amplification methods are required.

Isothermal methods of amplification (rapid amplification kinetics and efficiency) are especially useful for shorter oligonucleotides, including miRNAs; they have several advantages over methods based on the real-time polymerase chain reaction (RT-PCR), northern blotting, or micro arrays [9,10]. Such methods are less destructive toward biomacromolecules, retain the viability of recognition sites, allow variations in the photophysical properties of the fluorophores, and ensure the structural stability of the amplified nucleic acids during the entire process, including the isolation and separation steps [11]. Several isothermal DNA/RNA amplification methods have been developed previously: nucleic acid sequence–based amplification; strand displacement amplification; loop-mediated isothermal amplification; branched DNA, hybrid capture, and DNA cleavage–based signal amplification; the ligase chain reaction; and rolling circle amplification (RCA) [12]. RCA uses a circular template that is generally produced in the presence of the perfectly matched nucleic acid sequence and T4 ligase at ambient temperature. RCA techniques have been applied to identify DNA/RNA with excellent sensitivity (down to attomolar concentrations) and selectivity (1 in 10⁶) in complex mixtures.

In this study, we exploited the complexation and decomplexation processes of the hydrazone-based probe 1 (Scheme 1) upon the sequential additions of Cu²⁺ and pyrophosphate (PPi) ions to develop a simple and robust label-free method for the recognition of miRNAs using a convenient RCA process under modified buffer conditions. Several reports describe the identification of PPi ions during nucleic acid amplification processes, mainly relying on PCR [[13], [14], [15]]. Furthermore, various label-free methods have been developed for real-time monitoring using RCA [[16], [17], [18]]. Inspired by those studies, here we initiated a simple and robust label-free method for the recognition of miRNAs in a colorimetric and fluorimetric manner, along with their real-time monitoring.

To establish a simple and handy devise for the nucleic acid chemist to perform isothermal amplification, in this study we investigated some new chromo-fluorogenic techniques. Because of the great significance of miRNA-146a in genetics [19,20], we chose it as the prime target for the RCA process. Herein, we report the highly selective, sensitive, and robust chromo-fluorogenic Cu²⁺-based ensemble 1C for the detection of miRNA-146a, along with amplification monitoring with the aid of PPi (HP₂O₇³⁻) recognition (PPi produced during the RCA process) under modified ligation (T4 ligase)/polymerase (ϕ₂₉) buffer conditions through a switch-on response in the blueish-green (1) emission channel, as well as a red-to-colorless transition of the solution. We also describe a simple formulation for modification of the T4 ligation and ϕ₂₉ buffer to improve the sensitivity of miRNA detection in terms of the PPi produced through the enzymatic isothermal amplification process under physiological conditions. To the best of our knowledge, colorimetric and fluorimetric methods for miRNA-146a recognition, performed using a PPi sensing platform involving enzymatic isothermal amplification of nucleic acids under modified buffer conditions, have not been reported previously.

Section snippets

General materials and methods

¹H and ¹³C NMR spectra were recorded using a Bruker AM-400 spectrometer. UV–Vis absorption spectra were recorded using a Shimadzu UV-1650PC spectrophotometer. Fluorescence spectra were recorded using a JASCO FP-6500 fluorescence spectrometer (equipped with a Xe discharge lamp) and 1-cm quartz cells, with an excitation and emission slit width of 1/10 nm (readings obtained in 96-well plates). All optical measurements were performed at 25 °C. Solutions of the probe 1 were prepared, stored, and

Results and discussion

Considering the cost, the redox capabilities of divalent copper in the biological window, and the superiority of copper over other transition metals in terms of toxicity and biocompatibility [24], for this study we designed a highly selective Cu²⁺ ensemble (1C) capable of “switch-on” responses toward PPi. After photophysical studies of the excited states of various chromo-fluorescent materials, we found that only a few fluorophores with selected functionality exhibited the peculiar variations

miRNA sensing

Taking advantage of the sequential colorimetric and fluorimetric changes in the recognition of Cu²⁺ and PPi ions in MeCN/H₂O (3:7, v/v%) at pH 7.0 (HEPES buffer), we initiated isothermal amplification of miRNA using a circular template. During the enzymatic (ϕ₂₉ polymerase) amplification of miRNAs, PPi is generated through the consumption of dNTPs. Hence, we could track the enzymatically produced PPi analyte, recognized using a Cu²⁺ ensemble in modified ϕ₂₉ buffer, at pH 7.5 [40 mM Tris-HCl,

Conclusion

We have developed a highly sensitive and robust method for miRNA sensing, and for monitoring its signal amplification, based on a label-free approach using the chromo-fluorescent Cu²⁺ ensemble 1C. The sensing occurred through recognition of PPi ions produced during the signal amplification process, with selective extrusion of Cu²⁺ ions from the ensemble 1C resulting in the red color (of 1C) returning to colorless (for 1) and with fluorescence appearing in the blueish-green channels. We

Declaration of competing interest

Authors declare no conflicts of interest.

Acknowledgment

This study was supported by the National Research Foundation of the Republic of Korea (2017R1A2B4002398).

References (37)

B.H. Le et al.
Highly sensitive miRNA-146a detection using gold nanoparticle based probing system and isothermal amplification
Anal. Chim. Acta
(2018)
C.S. Zhu et al.
Avenues toward miRNA detections in vitro: a review of technical advances and challenges
Comput. Struct. Biotech. J.
(2019)
T. Tian et al.
A review: miRNA detection methods
Org. Biomol. Chem.
(2015)
M. de Planell-Saguer et al.
Detection methods of miRNAs in clinical practice
Clin. Biochem.
(2013)
B. Schweitzer et al.
Combined nucleic acid amplification and detection
Curr. Opin. Biotechnol.
(2001)
J. Qiang et al.
A dinuclear copper(II) complex based sensor for pyrophosphate and its applications to detecting pyrophosphatase activity and monitoring polymerase chain reaction
Sensors Actuators B Chem.
(2016)
Z. Li et al.
Di-nuclear Cu(II) complex bearing guanidinium arms for sensing pyrophosphate and monitoring the activity of PPase
Dyes Pigments
(2019)
H.-X. Jiang et al.
G-quadruplex fluorescent probe-mediated real-time rolling circle amplification strategy for highly sensitive microRNA detection
Anal. Chim. Acta
(2016)
R.P. Sartoris et al.
Structural and EPR studies of pyrophosphate-bridged dinuclear Cu^II complexes
Polyhedron
(2014)
A.S. Boutorine et al.
Fluorescent probes for nucleic acid visualization in fixed and live cells
Molecules
(2013)

D.-L. Ma et al.

Label-free luminescent oligonucleotide-probes

Chem. Soc. Rev.

(2013)

X. Miao et al.

Label-free platform for microRNA detection based on the fluorescence quenching of positively charged gold nanoparticles to silver nanoclusters

Anal. Chem.

(2018)

Y. Xia et al.

Recent advances in high-performance fluorescent and bioluminescent RNA imaging probes

Chem. Soc. Rev.

(2017)

Y. Cheng et al.

Recent advances in miRNA detection

Analyst

(2018)

M.M. Ali et al.

Chem. Soc. Rev.

(2014)

W. Li et al.

MicroRNA detection by microarray

Anal. Bioanal. Chem.

(2009)

B.K. Datta et al.

Zn²⁺ and pyrophosphate sensing: selective detection in physiological conditions and application in DNA-based estimation of bacterial cell numbers

Anal. Chem.

(2013)

H.-X. Jiang et al.

Real time monitoring of rolling circle amplification using aggregation induced emission, applications in biological detection

Chem. Commun.

(2015)

Cited by (17)

Advancements in visualizing loop-mediated isothermal amplification (LAMP) reactions: A comprehensive review of colorimetric and fluorometric detection strategies for precise diagnosis of infectious diseases
2024, Coordination Chemistry Reviews
In the context of global disease conditions and the associated mortality attributed to pathogens, nucleic acid amplification tests are crucial for precise and prompt diagnosis of infectious diseases. Among these tests, loop-mediated isothermal amplification (LAMP) has gained prominence due to its rapidity, simplicity, and high sensitivity. Visualizing the results of LAMP reactions is crucial for their application in diverse fields, and this comprehensive review explores recent advancements in colorimetric and fluorometric detection strategies. Beginning with an overview of global disease burdens and the significance of nucleic acid amplification tests, we delve into the LAMP reaction mechanism, focusing on deoxyribonucleic acid (DNA) polymerization and the associated by-products. Visualization of LAMP reactions can be categorized into indirect and direct detection methods. For indirect detection, we explore various techniques, including turbidity measurements and the use of colorimetric and fluorometric molecules for pH, Mg²⁺, and pyrophosphate (PPi) measurement. Techniques enabling direct detection of LAMP reactions are covered comprehensively, encompassing DNA-targeting colorimetric and fluorescent dyes, DNA hydrolysis, DNAzyme-based detection probes, Taqman probes, nanoparticles-based DNA hybridization probes, and the emerging role of nanozymes. Each of these methods offers unique advantages and applications in the visualization of LAMP reactions. This review provides a comprehensive overview of recent advances in detection strategies for LAMP reactions, shedding light on their potential in improving the diagnosis of infectious diseases and highlighting their versatility in various fields such as clinical diagnostics, environment monitoring, and food safety.
Novel Isoxazolylpyrimidine Derivatives: Design, Synthesis, Antifungal Activity and In-Silico Studies
2024, Asian Journal of Organic Chemistry
The advancement of environmentally friendly chemical synthesis in both academic and corporate research has recently taken on enormous relevance. Pesticides are substances, either chemical or biological, that are used to get rid of or lessen the impact of pests. To control the fungi in crops a new set of isoxazolylpyrimidine derivatives were planned, synthesized, and characterized. The compounds indicated were synthesized in high yields, and their structures were determined using elemental analysis, ¹H NMR, ¹³C NMR, and LCMS. HPLC was used to assess the purity of the synthesized molecules, and the purity of some compounds were found to be >98 % a/a while a couple were found to be >95 % a/a. The synthesized compounds were assessed for their antifungal activity. Among these, compounds 5d, 5h, and 5l exhibit substantial antifungal action and considerably suppress the growth of Rhizoctonia solani, Sclerotium rolfsii, and Macrophominaphaseolina. Macrophominaphaseolina inhibition percentages were 77.60, 70.49, 69.03, Sclerotium rolfsii inhibition percentages were 78.73, 74.03, 68.44, and Rhizoctonia solani inhibition percentages were 81.77, 76.29, and 71.01 for compounds 5 d, 5 h and 5 l respectively. The plausible mechanism of fungal inhibitory activities of rationally designed isoxazolyl pyrimidine derivatives is attributed to the blocking of the oxygen activation of HEME iron in membrane‐bound 14α‐demethylase. The presence of pyrimidine unit in designed materials is a vital factor for inducing antifungal activities, which was further corroborated through the density functional theory (DFT),molecular docking studies and molecular dynamics simulation studies.
Multiple ligation–Assisted recombinase polymerase amplification for highly sensitive and selective colorimetric detection of SARS-CoV-2
2023, Talanta
Citation Excerpt :
PK-probe is a Cu2+ complex of a thienyl-hydrazone rhodamine derivative. Several Cu2+-complex probes have been used in the colorimetric detection of DNA/RNA amplification [28]; even though they were selective to PPi, they underwent cross reactions with DTT [29]. Another type of AuNCs-Cu2+ system has been used to detect PPi in human urine [30].
In this paper we present a new method for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), targeting a specific region “N gene.” Under isothermal reaction conditions, we integrated ligation (Lig; high selectivity) and recombinase polymerase amplification (RPA; high sensitivity) processes, obtaining a robust method of detection. For point-of-care testing, we incorporated our laboratory-produced pyrophosphate ion (PPi)–sensing probe (PK-probe) for colorimetric analysis of the reaction. The total detection system was efficient and effective at diagnosing this RNA virus–mediated disease rapidly (30 min). In a full-genome SARS-CoV-2 study, our PK-probe/Lig-RPA system functioned with a limit of detection of 1160 copies/ml, with a single-mismatch level of selectively, and it was highly selective even in the presence of bacterial genomes commonly found in the human mouth and nose. This robust, straightforward, selective, efficient, and ultrasensitive colorimetric detection method, with potential for point-of-care analysis, should also be effective in detecting a diverse range of other RNA-based diseases.
An efficient p-TSA catalyzed synthesis of some new substituted-(5-hydroxy-3-phenylisoxazol-4-yl)-1,3-dimethyl-1H-chromeno[2,3-d]pyrimidine-2,4(3H,5H)-dione/3,3-dimethyl-2H-xanthen-1(9H)-one scaffolds and evaluation of their pharmacological and computational investigations
2022, Journal of Molecular Structure
Citation Excerpt :
Further, the Frontier Molecular Orbital (FMO) analysis can provide important information about electronic properties, chemical reactivity, biological activity, and kinetic stability of the molecules [31]. The HOMO and LUMO of compounds were helpful in determining various global reactivity parameters, donor-acceptor interaction, and intramolecular charge transfer (ICT) ability of compounds [32,33]. In this avenue, the current study aims to synthesize novel substituted-(5-hydroxy-3-phenylisoxazol-4-yl)-1,3-dimethyl-1H-chromeno[2,3-d]pyrimidine-2,4(3H,5H)-dione/3,3-dimethyl-2H-xanthen-1(9H)-one derivatives and evaluate some of their biological properties, such as cytotoxicity and anti-TB activity.
We have developed an easy and efficient protocol for the synthesis of a series of some novel substituted-(5-hydroxy-3-phenylisoxazol-4-yl)-1,3-dimethyl-1H-chromeno[2,3-d]pyrimidine-2,4(3H,5H)-dione/3,3-dimethyl-2H-xanthen-1(9H)-one derivatives (4a–j) via p-TSA catalyzed reaction of 1,3-dimethylbarbituric acid/dimedone, substituted salicylaldehyde/2-hydroxy-nepthaldehyde and 3-phenyl-5-isoxazolone was administered in refluxed temperature in the presence of aqueous ethanol. All the obtained compounds were evaluated for their therapeutic effect using pharmacological and computational investigations, and structures were confirmed using analytical and spectroscopic techniques. Absorption spectra were recorded in six different solvents such as polar protic, polar aprotic, and nonpolar solvents, λ_max of all the compounds are appeared at bathochromic shift towards the longer wavelength due to the π-π* & n-π* transitions. The results of pharmacological investigations revealed that the compounds 4c, 4e, and 4h possess excellent cytotoxicity efficacy, and compounds 4c, 4d, 4h, and 4i have shown better anti-TB efficiency. The SAR study shows the importance of the electron-withdrawing group and additional phenyl nucleus enhancing the biological potency of the compounds. The results of the in silico molecular docking studies revealed that the compounds 4c and 4h effectively interacted with P38 MAP kinase (-10.9 kcal/mol) and InhA-Enoyl-Acyl Carrier Protein Reductase (-11.0 kcal/mol), respectively. Further, the molecular dynamics (MD) simulation studies revealed that the compounds 4c and 4h stabilized the macromolecular structures in terms of RMSD, RMSF, the Radius of gyration, and SASA compared to their unbound and standard compound bound structures. DFT study suggested that the compounds are chemically and biologically more reactive due to less energy gap.
A dimedone-phenylalanine-based fluorescent sensor for the detection of iron (III), copper (II), L-cysteine, and L-tryptophan in solution and pharmaceutical samples
2022, Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy
The development of fluorescence molecules for the fast and effective detection of L-tryptophan (L-Trp) has attracted a lot of attention because it is an important amino acid for baby growth, nitrogen equilibrium in adults, improving sleep, and mood regulation. A dimedone-phenylalanine-based chiral sensor (SDPA) was synthesized and exhibited a strong fluorescence quenching by Fe³⁺ and Cu²⁺ in a water/DMSO (3/7) solution with a detection limit of 2.29 × 10^-6 M and 6.37 × 10^-6 M, respectively. The factors affecting fluorescence sensings, such as the pH and competing cations, were studied. The sensor can be reused at least five times after being treated with EDTA. The Job plot, ESI-MS spectra, ¹H NMR spectra, absorbance, and fluorescence titration experiments were investigated to study the mechanism of SDPA-Fe³⁺ and SDPA-Cu²⁺ complexation. The SDPA-Cu²⁺ complex can detect L-tryptophan and L-cysteine at trace levels by turn-on fluorescence with a detection limit of 9.35 × 10^-6 M and 8.86 × 10^-6 M, respectively. Moreover, applying the SDPA-Cu²⁺ complex for quantitative analysis of L-tryptophan in real sleep-improving capsules resulted in good recovery. The L-tryptophan level of the Elining capsule was determined at 190.8 ± 10.5 mg/g (mg L-tryptophan/g medicine), which is close to the announced quantity of 180 mg/g. Besides, the SDPA-Cu²⁺ complex can selectively detect free L-Try molecules and L-Try residues in proteins.
Copper complex of a thienyl-hydrazone rhodamine derivative is a highly selective colorimetric sensor for pyrophosphate
2022, Tetrahedron Letters
Citation Excerpt :
Several fluorogenic PPi-sensing complex probes have been reported, particularly those targeting the polymerase chain reaction, but they have not been selective for PPi in the presence of other competing anionic molecules [11–13]. Furthermore, various strategies have been used to detect PPi units, including displacement assays [14–17], aggregation-induced emission and quenching [18–20], chemical reactions [21–27], and indicator displacement assays [28,29]. Many of these probes are, however, poorly resistant to other biomolecules and buffer components, making them nonselective for PPi.
Detection of pyrophosphate (PPi) can be used to monitor gene replication, transcription, and other forms of enzymatic DNA/RNA synthesis, but it can be difficult to perform in real samples because of the presence of many other biomolecules and buffer components. In this study we prepared a colorimetric probe that could detect PPi specifically and selectively in the presence of cysteine, dithiothreitol (DTT), nucleoside triphosphates, and human serum (containing several biomolecules and amino acids). Among four candidate rhodamine derivatives, we found that the copper complex 1 could detect PPi uniquely, even in the presence of several competing biomolecules. The copper complex 1 also exhibited its highly selective PPi sensing capability when applied to rolling circle amplification and in vitro transcription in samples containing high concentrations of DTT, making it a promising candidate for colorimetric detection of RNA/DNA when using amplification techniques.

View all citing articles on Scopus

View full text

Label-free sensing platform for miRNA-146a based on chromo-fluorogenic pyrophosphate recognition

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

General materials and methods

Results and discussion

miRNA sensing

Conclusion

Declaration of competing interest

Acknowledgment

Anal. Chim. Acta

Comput. Struct. Biotech. J.

Org. Biomol. Chem.

Clin. Biochem.

Curr. Opin. Biotechnol.

Sensors Actuators B Chem.

Dyes Pigments

Anal. Chim. Acta

Polyhedron

Fluorescent probes for nucleic acid visualization in fixed and live cells

Molecules

Label-free luminescent oligonucleotide-probes

Chem. Soc. Rev.

Label-free platform for microRNA detection based on the fluorescence quenching of positively charged gold nanoparticles to silver nanoclusters

Anal. Chem.

Recent advances in high-performance fluorescent and bioluminescent RNA imaging probes

Chem. Soc. Rev.

Recent advances in miRNA detection

Analyst

Chem. Soc. Rev.

MicroRNA detection by microarray

Anal. Bioanal. Chem.

Zn2+ and pyrophosphate sensing: selective detection in physiological conditions and application in DNA-based estimation of bacterial cell numbers

Anal. Chem.

Real time monitoring of rolling circle amplification using aggregation induced emission, applications in biological detection

Chem. Commun.

Zn²⁺ and pyrophosphate sensing: selective detection in physiological conditions and application in DNA-based estimation of bacterial cell numbers