Dimeric G-quadruplex Research Articles

Cu2+-mediated telomeric dimeric G-quadruplex DNAzyme for highly sensitive colorimetric detection of deferasirox

Deferasirox (DEF) is an important iron chelator for treatment of iron overload–related diseases. Monitoring DEF concentration in human serum will provide some valuable information for clinical diagnosis and therapy of such diseases. In this study, we developed a peroxidase-mimicking colorimetric sensor for the detection of DEF by simple assembly of a telomeric dimeric G-quadruplex DNAzyme with Cu2+. The DNAzyme–catalyzed oxidation of 3,3′,5,5′-tetramethylbenzidine in the presence of H2O2 can generate a quantitative colorimetric signal, and the color change can be discerned by the naked eye. Compared with the reaction rate of the monomeric G-quadruplex–Cu2+ DNAzyme, the reaction rate of the dimeric G-quadruplex–Cu2+ (G2–Cu2+) DNAzyme is significantly accelerated, and the reaction rate gradually increases and then reaches a plateau with increasing number of TTA spacers. Herein, the G2–Cu2+ DNAzyme is chosen for the highly sensitive detection of DEF based on the DEF–Cu2+ complex–induced inhibition of its peroxidase-mimicking activities. The limit of detection (LOD) of DEF is achieved as low as 0.03 μM, and the linear range is from 0.05 to 1.2 μM. The proposed strategy exhibits excellent selectivity in the presence of potential interferents, such as metal ions and small molecules. Importantly, the G2–Cu2+ DNAzyme is further expanded to detect DEF in dispersible tablets and human serum samples. Overall, this G2–Cu2+ DNAzyme provides a simple, low-cost, and rapid platform for DEF detection. This novel strategy is the first example of DEF analysis by utilizing signal amplification technology based on the G-quadruplex DNAzyme and holds great potential for DEF quality control and therapeutic drug monitoring.

Crystal structure of a tetrameric RNA G-quadruplex formed by hexanucleotide repeat expansions of C9orf72 in ALS/FTD.

The abnormal GGGGCC hexanucleotide repeat expansions (HREs) in C9orf72 cause the fatal neurodegenerative diseases including amyotrophic lateral sclerosis and frontotemporal dementia. The transcribed RNA HREs, short for r(G4C2)n, can form toxic RNA foci which sequestrate RNA binding proteins and impair RNA processing, ultimately leading to neurodegeneration. Here, we determined the crystal structure of r(G4C2)2, which folds into a parallel tetrameric G-quadruplex composed of two four-layer dimeric G-quadruplex via 5'-to-5' stacking in coordination with a K+ ion. Notably, the two C bases locate at 3'- end stack on the outer G-tetrad with the assistance of two additional K+ ions. The high-resolution structure reported here lays a foundation in understanding the mechanism of neurological toxicity of RNA HREs. Furthermore, the atomic details provide a structural basis for the development of potential therapeutic agents against the fatal neurodegenerative diseases ALS/FTD.

Engineering of a DNAzyme-Based dimeric G-quadruplex rolling circle amplification for robust analysis of lead ion

Detecting heavy metal pollution, particularly lead ion (Pb2⁺) contamination, is imperative for safeguarding public health. In this study, we introduced an innovative approach by integrating DNAzyme with rolling circle amplification (RCA) to propose an amplification sensing method termed DNAzyme-based dimeric-G-quadruplex (dimer-G4) RCA. This sensing approach allows for precise and high-fidelity Pb2⁺ detection. Strategically, in the presence of Pb2⁺, the DNAzyme undergoes substrate strand (S-DNA) cleavage, liberating its enzyme strand (E-DNA) to prime isothermal amplification. This initiates the RCA process, producing numerous dimer-G-Quadruplexes (dimer-G4) as the signal reporting transducers. Compared to conventional strategies using monomeric G-quadruplex (mono-G4) as the reporting transducers, these dimer-G4 structures exhibit significantly enhanced fluorescence when bound with Thioflavin T (ThT), offering superior target signaling ability for even detection of Pb2⁺ at low concentration. Conversely, in the absence of Pb2⁺, the DNAzyme structure remains intact so that no primers can be produced to cause the RCA initiation. This nucleic acid amplification-based Pb2⁺ detection method combing with the high specificity of DNAzymes for Pb2⁺ recognition ensures highly sensitive detection of Pb2+ with a detection limit of 0.058 nM, providing a robust tool for food safety analysis and environmental monitoring.

Sensitive fluorescence detection based on dimeric G-quadruplex combined with enzyme-assisted solid-phase microextraction of streptomycin in honey

Streptomycin (STR), an aminoglycoside antibiotic with the potential to persist in honey and other food products, may induce allergy, toxicity and antibiotic resistance in humans. In this study, we developed a solid-phase microextraction (SPME) biosensor based on a quartz rod that was modified with double-stranded DNA structures consisting of partially complementary G-rich base DNA strand and STR aptamer. The STR isolated by SPME initially bound to the aptamer. Then the remaining double-stranded DNA structures were cleaved by the Nt.BstNBI enzyme, resulting in release of G-quadruplex dimers. The latter formed a complex with thioflain T fluorescent dye, resulting in an amplified fluorescence response. The method exhibited high sensitivity (a limit of detection of 10.84 pM), wide linear range (0.05 nM ∼ 500 nM (with determination coefficient > 0.99)), and simple operation, making it suitable and convenient for STR detection. Successful STR determination in genuine honey samples was demonstrated.

CRISPR/Cas12a cleavage-mediated isothermal amplification lights up the dimeric G-quadruplex signal unit for ultrasensitive and label-free detection of circulating tumor DNA

Circulating tumor DNA (ctDNA) is a valuable and potential cancer biomarker for real-time monitoring, and plays a vital role in cancer early diagnosis. In this work, we developed an ultrasensitive and label-free fluorescent biosensor for ctDNA detection. A well-designed RNA-DNA chimeric primer (RD) with phosphate groups modified at the 3-terminal was cleaved by target-initiated CRISPR/Cas12a, and then the subsequent exponential amplification reaction (EXPAR) was started to produce numerous G-rich sequences, which could be assembled with Thioflavin T to form a dimeric G-quadruplex (dimer-G4/ThT) and significantly enhance the fluorescence. Due to the innovative combination of the specificity of CRISPR/Cas12a and RD primers, the high amplification efficiency of EXPAR, and the strong fluorescence response of dimer-G4/ThT, this strategy demonstrated excellent sensitivity at the aM level. In addition, the sensing system showed satisfactory anti-interference ability and reliable accuracy in complex serum environment and tumor cell lysate. More importantly, this strategy avoided the complicated and expensive labeling process of traditional signal probes, making the construction of fluorescent biosensor simpler and more practical, offering practical insights for molecular diagnostics and clinical applications.

A novel self-feedback biosensor for label-free detection of APE1 by primer exchange reaction and rolling circle amplification/dimeric G-quadruplex

Human apurinic/apyrimidinic endonuclease 1(APE1), a critical DNA base excision repair enzyme, plays a fundamental role in nucleotide damage repair and transcriptional regulation, emerging as a vital target for anticancer therapy. Therefore, sensitive and label-free detection of APE1 is necessary. Herein, a primer exchange reaction (PER) cascade rolling circle amplification (RCA)/ dimeric G-quadruplex (G4) self-feedback biosensor was developed to detect APE1. Initially, a gated hairpin (GHP) was meticulously crafted for the specific recognition of APE1, in which GHP is not only the recognition substrate but also acts as a catalytic hairpin (CHP) and a primer for PER. The target recognizes and cleaves the GHP, instigating the PER cycle and engendering extended single-strands DNA, thereby activating RCA reaction. Subsequently, the resulting protracted tandem G-rich ssDNA conformationally adapts into a pronounced G4 dimeric structure through affinity binding to K+ and thioxlavin T (ThT), culminating in a stronger fluorescence signal. The design of GHP and G4 dimer greatly improves the signal-to-noise ratio and detection sensitivity, with a wide linear range (0.02–30 U/mL) and a low limit of detection (0.001 U/mL). Moreover, this protocol exhibits robust resistance to interference and holds significant potential in pharmaceutical evaluation, which may provide a reliable analytical method for the detection of APE1 and the subsequent clinical assessment of associated malignancies.

Parallel G-quadruplex recognition by neomycin.

G-quadruplex-forming nucleic acids have evolved to have applications in biology, drug design, sensing, and nanotechnology, to name a few. Together with the structural understanding, several attempts have been made to discover and design new classes of chemical agents that target these structures in the hope of using them as future therapeutics. Here, we report the binding of aminoglycosides, in particular neomycin, to parallel G-quadruplexes that exist as G-quadruplex monomers, dimers, or compounds that have the propensity to form dimeric G-quadruplex structures. Using a combination of calorimetric and spectroscopic studies, we show that neomycin binds to the parallel G-quadruplex with affinities in the range of Ka ∼ 105-108M-1, which depends on the base composition, ability to form dimeric G-quadruplex structures, salt, and pH of the buffer used. At pH 7.0, the binding of neomycin was found to be electrostatically driven potentially through the formation of ion pairs formed with the quadruplex. Lowering the pH resulted in neomycin's association constants in the range of Ka ∼ 106-107M-1 in a salt dependent manner. Circular dichroism (CD) studies showed that neomycin's binding does not cause a change in the parallel conformation of the G-quadruplex, yet some binding-induced changes in the intensity of the CD signals were seen. A comparative binding study of neomycin and paromomycin using d(UG4T) showed paromomycin binding to be much weaker than neomycin, highlighting the importance of ring I in the recognition process. In toto, our results expanded the binding landscape of aminoglycosides where parallel G-quadruplexes have been discovered as one of the high-affinity sites. These results may offer a new understanding of some of the undesirable functions of aminoglycosides and help in the design of aminoglycoside-based G-quadruplex binders of high affinity.

Dimeric Metal-Salphen Complexes Which Target Multimeric G-Quadruplex DNA

G-Quadruplex DNA structures have attracted increasing attention due to their biological roles and potential as targets for the development of new drugs. While most guanine-rich sequences in the genome have the potential to form monomeric G-quadruplexes, certain sequences have enough guanine-tracks to give rise to multimeric quadruplexes. One of these sequences is the human telomere where tandem repeats of TTAGGG can lead to the formation of two or more adjacent G-quadruplexes. Herein we report on the modular synthesis via click chemistry of dimeric metal-salphen complexes (with NiII and PtII) bridged by either polyether or peptide linkers. We show by circular dichroism (CD) spectroscopy that they generally have higher selectivity for dimeric vs monomeric G-quadruplexes. The emissive properties of the PtII-salphen dimeric complexes have been used to study their interactions with monomeric and dimeric G-quadruplexes in vitro as well as to study their cellular uptake and localization.

Unusual topological RNA G-quadruplex formed by an RNA duplex: implications for the dimerization of SARS-CoV-2 RNA.

The infectious disease coronavirus 2019 (SARS-CoV-2) is caused by a virus that has RNA as its genetic material. To understand the detailed structural features of SARS-COV-2 RNA, we probed the RNA structure by NMR. Two RNA sequences form a duplex and self-associate to form a dimeric G-quadruplex. The FrG nucleoside was employed as a 19F sensor to confirm the RNA structure in cells by 19F NMR. A FRET assay further demonstrated that the dimeric G-quadruplex resulted in RNA dimerization in cells. These results provide the basis for the elucidation of SARS-COV-2 RNA function, which provides new insights into developing novel antiviral drugs against SARS-COV-2.

Selective light-up of dimeric G-quadruplex forming aptamers for efficient VEGF165 detection

To develop efficient anticancer theranostic systems, we studied the interaction between a cyanine dye, analogue of thiazole orange (named CyOH), and two G-quadruplex-forming aptamers, V7t1 and 3R02, recognizing the Vascular Endothelial Growth Factor 165 (VEGF165) - an angiogenic protein overexpressed in cancer cells, responsible for the rapid growth and metastases of solid tumours. We demonstrated, by exploiting different biophysical techniques - i.e. gel electrophoresis, circular dichroism (CD), UV–vis and fluorescence spectroscopy - that this cyanine interacted with both aptamers giving a marked fluorescence light-up only when bound to their dimeric forms. Interestingly, both oligonucleotides recognized VEGF165 with higher affinity when adopting dimeric G-quadruplexes, largely prevalent over their monomeric forms in pseudo-physiological conditions. Notably, the fluorescence light-up produced by the probe was maintained when the dimeric aptamer-CyOH complexes bound to the target protein. These complexes, tested on MCF-7 cancer cells using non-tumorigenic MCF-10A cells as control, were effectively internalized in cells and colocalized with a fluorescently-labelled anti-VEGF-A antibody, allowing both recognition and detection of the target. Our experiments showed that the studied systems are promising tools for anticancer theranostic strategies, combining the therapeutic potential of the G4-forming anti-VEGF aptamers with the diagnostic efficacy of the cyanine selective fluorescence light-up.

A label-free T4 polynucleotide kinase fluorescence sensor based on split dimeric G-quadruplex and ligation-induced dimeric G-quadruplex/thioflavin T conformation

The phosphorylation process of DNA by T4 polynucleotide kinase (T4 PNK) plays a crucial role in DNA recombination, DNA replication, and DNA repair. Traditional monomeric G-quadruplex (G4) systems are always activated by single cation such as K+ or Na+. The conformation transformation caused by the coexistence of multiple cations may interfere with the signal readout and limit their applications in physiological system. In view of the stability of dimeric G4 in multiple cation solution, we reported a label-free T4 PNK fluorescence sensor based on split dimeric G4 and ligation-induced dimeric G4/thioflavin T (ThT) conformation. The dimeric G4 was divided into two independent pieces of one normal monomeric G4 and the other monomeric G4 fragment phosphorylated by T4 PNK in order to decrease the background signal. With the introduction of template DNA, DNA ligase, and invasive DNA, the dimeric G4 could be generated and liberated to combine with ThT to show obvious fluorescence signal. Using our strategy, the linear range from 0.005 to 0.5 U mL-1, and the detection limit of 0.0021 U mL-1 could be achieved without the consideration of interference caused by the coexistence of multiple cations. Additionally, research in real sample determination and inhibition effect investigations indicated its further potential application value in biochemical process research and clinic diagnostics.

The parallel tetrameric DNA G-quadruplex formed by the two-repeat C9orf72 GGGGCC sequence in solution

The abnormal expansion of G-rich hexanucleotide repeat, GGGGCC (G4C2), in chromosome 9 open reading frame 72 (C9orf72) is known to be the prevailing genetic cause of two fatal degenerative neurological diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). It is well known that the DNA G4C2 repeat expansion with different lengths can form G-quadruplexes which affect gene transcription related to ALS/FTD, therefore it is crucial to understand DNA G4C2 G-quadruplex structures. Herein, by utilizing nuclear magnetic resonance (NMR) spectroscopy, we examined DNA G-quadruplex structure adopted by two G4C2 hexanucleotide repeats with an inosine substitution at position 4, d(G4C2)2-I4. We show that d(G4C2)2-I4 folds into an eight-layer parallel tetrameric G-quadruplex containing two parallel dimeric G-quadruplexes stacking together through π-π interaction via 5′-to-5′ mode in solution. Each dimeric G-quadruplex unit involves two propeller loops composed of two cytosine bases. This result is consistent with the observation in the crystal structure of d(G4C2)2. Our work not only sheds light on the structural diversity of G-quadruplexes adopted by d(G4C2)n but also provides a structural basis for drug design in treatment of ALS and FTD.

NIR imaging human telomeric dimeric G-quadruplexes by malachite green dimers and their antitumor activity

Though a small number of binders and probes towards human telomeric multimeric G-quadruplexes have been reported to use the tumor detection and therapy in recent years, their detection in visible region and medium antitumor activity limit the development in the research of antitumor drugs. Here, three new malachite green (MG) dimers 1a∼c with the different-length polyether linkers have been synthesized to study their NIR visualization towards human telomeric dimeric G-quadruplexes (G2T1) and antitumor activity. The linkers of these dimers could optimize the imaging and binding selectivity for G2T1. Dimer 1c with the longest polyether linker showed higher binding and stabilization towards G2T1 over monomeric G-quadruplexes and ds DNA. More importantly, dimer 1c exhibited selective NIR imaging towards G2T1 in vitro and in cells. In addition, dimer 1c also showed excellent cytotoxicity (IC50 < 1.0 μM). This study is one of the few research reports on G2T1 probes combined with NIR imaging and the excellent antitumor activity. It gives us to provide a new idea to develop the antitumor drugs with high efficiency and the less side effect by controlling the drug delivery under the guidance of NIR imaging towards G2T1.

Crystal structures of an HIV-1 integrase aptamer: Formation of a water-mediated A•G•G•G•G pentad in an interlocked G-quadruplex

93del is a 16-nucleotide G-quadruplex-forming aptamer which can inhibit the activity of the HIV-1 integrase enzyme at nanomolar concentration. Previous structural analyses of 93del using NMR spectroscopy have shown that the aptamer forms an interlocked G-quadruplex structure in K+ solution. Due to its exceptional stability and unique topology, 93del has been used in many different studies involving DNA G-quadruplexes, such as DNA aptamer and multimer design, as well as DNA fluorescence research. To gain further insights on the structure of this unique aptamer, we have determined several high-resolution crystal structures of 93del and its variants. While confirming the overall dimeric interlocked G-quadruplex folding topology previously determined by NMR, our results reveal important detailed structural information, particularly the formation of a water-mediated A•G•G•G•G pentad. These insights allow us to better understand the formation of various structural elements in G-quadruplexes and should be useful for designing and manipulating G-quadruplex scaffolds with desired properties.

Design of a High-Sensitivity Dimeric G-Quadruplex/Hemin DNAzyme Biosensor for Norovirus Detection.

G-quadruplexes can bind with hemin to form peroxidase-like DNAzymes that are widely used in the design of biosensors. However, the catalytic activity of G-quadruplex/hemin DNAzyme is relatively low compared with natural peroxidase, which hampers its sensitivity and, thus, its application in the detection of nucleic acids. In this study, we developed a high-sensitivity biosensor targeting norovirus nucleic acids through rationally introducing a dimeric G-quadruplex structure into the DNAzyme. In this strategy, two separate molecular beacons each having a G-quadruplex-forming sequence embedded in the stem structure are brought together through hybridization with a target DNA strand, and thus forms a three-way junction architecture and allows a dimeric G-quadruplex to form, which, upon binding with hemin, has a synergistic enhancement of catalytic activities. This provides a high-sensitivity colorimetric readout by the catalyzing H2O2-mediated oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline -6-sulfonic acid) diammonium salt (ABTS). Up to 10 nM of target DNA can be detected through colorimetric observation with the naked eye using our strategy. Hence, our approach provides a non-amplifying, non-labeling, simple-operating, cost-effective colorimetric biosensing method for target nucleic acids, such as norovirus-conserved sequence detection, and highlights the further implication of higher-order multimerized G-quadruplex structures in the design of high-sensitivity biosensors.

Crystal structure of parallel G-quadruplex formed by the two-repeat ALS- and FTD-related GGGGCC sequence.

The hexanucleotide repeat expansion, GGGGCC (G4C2), within the first intron of the C9orf72 gene is known to be the most common genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The G4C2 repeat expansions, either DNA or RNA, are able to form G-quadruplexes which induce toxicity leading to ALS/FTD. Herein, we report a novel crystal structure of d(G4C2)2 that self-associates to form an eight-layer parallel tetrameric G-quadruplex. Two d(G4C2)2 associate together as a parallel dimeric G-quadruplex which folds into a tetramer via 5′-to-5′ arrangements. Each dimer consists of four G-tetrads connected by two CC propeller loops. Especially, the 3′-end cytosines protrude out and form C·C+•C·C+/ C·C•C·C+ quadruple base pair or C•C·C+ triple base pair stacking on the dimeric block. Our work sheds light on the G-quadruplexes adopted by d(G4C2) and yields the invaluable structural details for the development of small molecules to tackle neurodegenerative diseases, ALS and FTD.

A highly selective switch-on fluorescence sensor targeting telomeric dimeric G-quadruplex

The fluorescence probes with high selectivity and sensitivity for telomeric multimeric G-quadruplexes have attracted much attention. Nevertheless, few small molecules have exhibited telomeric multimeric G-quadruplexes recognition specificity. Thus, there is an urgent demand to develop specific fluorescence probes for telomeric multimeric G-quadruplexes. We reported herein the specific sensing of telomeric dimeric G-quadruplex TTA45 via a fluorescence light-up response using a commercially available triazine derivative HPTA-1 as a probe. HPTA-1 could discriminate the telomeric dimeric G-quadruplex TTA45 against other types of DNA structures accompanied by a drastic enhancement of the emission intensity without compromising the conformation and stability. Compared with most multimeric G-quadruplex recognition ligands, HPTA-1 had much simpler structure and lower molecular weight. The binding mechanism studies suggested that the distinct fluorescence response was caused by electrostatic and π-π stacking interactions of HPTA-1 with the pocket between two G-quadruplex units of telomeric dimeric G-quadruplex TTA45..

Structural and functional characterization of complexes between heme and dimeric parallel G-quadruplex DNAs

Heme has been receiving considerable interest as a prosthetic group of ribozymes and deoxyribozymes, because heme-bound nucleic acids exhibit peroxidase-like catalytic activities (Travascio, P., Li, Y., and Sen, D. (1998) Chem. Biol, 5, 505–517). The interaction of heme with dimeric G-quadruplexes formed from d(TAGGGTTAGGGT) and d(TAGGGTTAGGGA) has been characterized to gain a deeper understanding of the molecular recognition of G-quadruplex DNAs by heme. We found that heme binds selectively to the 3′-terminal G-quartet of a dimeric parallel G-quadruplex of d(TAGGGTTAGGGT), whereas binding of heme to a dimeric antiparallel G-quadruplex of d(TAGGGTTAGGGA) does not occur, suggesting that an orderly arrangement of the constituent guanine deoxyribose rings, with respect to the G-quartet plane, is crucial for the binding of heme to the DNA. The preferential binding of heme to the 3′-terminal G-quartet of parallel G-quadruplex DNAs allowed a systematic modification of the heme environment in the complex through the DNA sequence. The activity of the complexes was found to increase with increasing number of adenine bases adjacent to the heme in the complexes, possibly due to improvement of the accessibility of aromatic substrate, i.e., 10-acetyl-3,7-dihydroxyphenoxazine, to the heme, and an increase in the frequency of appearance of a specific orientation of the adenine bases, with respect to the heme, optimized for its activity as an acid-base catalyst to enhance the peroxidase activity of the complex.

BMPQ-1 binds selectively to (3+1) hybrid topologies in human telomeric G-quadruplex multimers.

A single G-quadruplex forming sequence from the human telomere can adopt six distinct topologies that are inter-convertible under physiological conditions. This presents challenges to design ligands that show selectivity and specificity towards a particular conformation. Additional complexity is introduced in differentiating multimeric G-quadruplexes over monomeric species, which would be able to form in the single-stranded 3′ ends of telomeres. A few ligands have been reported that bind to dimeric quadruplexes, but their preclinical pharmacological evaluation is limited. Using multidisciplinary approaches, we identified a novel quinoline core ligand, BMPQ-1, which bound to human telomeric G-quadruplex multimers over monomeric G-quadruplexes with high selectivity, and induced the formation of G-quadruplex DNA along with the related DNA damage response at the telomere. BMPQ-1 reduced tumor cell proliferation with an IC50 of ∼1.0 μM and decreased tumor growth rate in mouse by half. Biophysical analysis using smFRET identified a mixture of multiple conformations coexisting for dimeric G-quadruplexes in solution. Here, we showed that the titration of BMPQ-1 shifted the conformational ensemble of multimeric G-quadruplexes towards (3+1) hybrid-2 topology, which became more pronounced as further G-quadruplex units are added.

Probing the Importance of the G-Quadruplex Grooves for the Activity of the Anti-HIV-Integrase Aptamer T30923.

In this paper, we report studies concerning four variants of the G-quadruplex forming anti-HIV-integrase aptamer T30923, in which specific 2′-deoxyguanosines have been singly replaced by 8-methyl-2′-deoxyguanosine residues, with the aim to exploit the methyl group positioned in the G-quadruplex grooves as a steric probe to investigate the interaction aptamer/target. Although, the various modified aptamers differ in the localization of the methyl group, NMR, circular dichroism (CD), electrophoretic and molecular modeling data suggest that all of them preserve the ability to fold in a stable dimeric parallel G-quadruplex complex resembling that of their natural counterpart T30923. However, the biological data have shown that the T30923 variants are characterized by different efficiencies in inhibiting the HIV-integrase, thus suggesting the involvement of the G-quadruplex grooves in the aptamer/target interaction.