Aptamer AS411 interacts with the KRAS promoter/hnRNP A1 complex and shows increased potency against drug-resistant lung cancer

Abstract

G-quadruplex (G4) aptamers that can competitively binding protein with oncogene promoter G4 hold promise for cancer treatment. In this study, a neutral cytidinyl lipid, DNCA, was shown to transfect and deliver G4 aptamers (AS1411, TBA) into tumour cells, including multidrug-resistant tumour cells, and their nuclear localizations were clearly detected. Both AS1411/DNCA and TBA/DNCA showed excellent antitumour efficacies in the drug-resistant non-small cell lung cancer cell line A549/TXL at a low concentration (100 nM). Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) was identified as a new target of AS1411 and TBA. The binding affinities were measured, and the K_d values of AS1411/hnRNP A1 and TBA/hnRNP A1 were 17.5 nM and 21.1 nM, respectively. Then the expression of KRAS mRNA in A549/TXL cells was found to be higher than that in A549 cells, and KRAS mRNA was reduced by approximately 40% after administration of AS1411 or TBA in A549/TXL cells. Further, it was confirmed for the first time that AS1411 targeted not only hnRNP A1 but also the KRAS promoter/hnRNP A1 complexes. And although TBA cannot target the KRAS promoter/hnRNP A1 complexes, the biolayer interferometry (BLI) experiment showed that TBA and AS1411 have similar effects on several key proteins in tumour cells, especially hnRNP A1. Molecular docking and molecular dynamics simulation showed that AS1411 and the KRAS promoter bound to the same domain of hnRNP A1 protein, while TBA bound to another domain.

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Introduction

Aptamers are short, single-stranded DNA or RNA ligands that can fold into unique three-dimensional structures to bind targets through spatial complementarities and intermolecular interactions.¹ Guanine-rich oligonucleotides (GRO) comprise many aptamers with the ability to form G4 structures. G4s are stabilized by the stacking of G-quartets, in which four guanines are assembled in a planar arrangement by Hoogsteen hydrogen bonding. The G4 structure can be formed by one, two or more DNA/RNA strands, and the directions of the strands can be parallel, antiparallel, or hybrid. Moreover, the G4 structure can vary in sequence and loop size.²

G4 structures in oncogene promoters, such as KRAS, BCL2, VEGF, and PDGFR-β, are functionally important and have emerged as a promising new class of antitumour targets. Large aromatic or cyclic compounds were considered the optimal G4 ligands, but a more recent study suggested that smaller asymmetric compounds with appropriate functional groups were better choices to specifically bind G4 structures.³ The NMR solution structures of the complexes of KRAS-G4 with berberine and the complexes of KRAS-G4 with coptisine, were determined respectively,⁴ and the complex structures were similar to the complex structures of berberine binding to a MYC-G4 or a dGMP-fill-in PDGFR-β vacancy G4.^5,6 Another strategy is to target proteins that interact with endogenous G4 structures, such as the heterogeneous nuclear ribonucleoprotein family (hnRNPs). hnRNPs are the most critical alternative splicing regulators, and they have been reported to be related to various aspects of cancer. The protein hnRNP A1, which is upregulated in a variety of cancers, unfolds the G4 of the K-Ras proto-oncogene (KRAS) promoter to promote its expression⁷ and binds to the G4 of the 5′-untranslated region of tyrosine kinase receptor RON mRNA to activate its translation.⁸ In some mechanistic studies, aptamers have been shown to compete with endogenous DNA or RNA to inhibit the formation of endogenous nucleic acid/protein complexes.⁹ For example, an anti-PCNA aptamer was developed by systematic evolution of ligands by exponential enrichment (SELEX) to compete with primer-template DNA for binding to the PCNA/DNA polymerase δ or ε complex.¹⁰ A linear L-RNA aptamer and a cyclized L-RNA aptamer targeting human telomerase RNA G-quadruplex (hTERC D-rG4) have been developed, and their inhibition of hTERC rG4–nucleolin interactions has been demonstrated.¹¹

As the most widely reported antitumour G4 aptamers, TBA and AS1411 attracted our attention. TBA (5′-GGT TGG TGT GGT TGG-3′) is a 15-nt aptamer obtained by SELEX screening that forms a chair-like antiparallel G4 structure. TBA can inhibit blood coagulation by specific binding with thrombin exosite I.¹² On the other hand, dibenzyl linker¹³ and 3′-5′-inversion combined with L-nucleoside¹⁴ modification at the loop region of TBA was reported to be able to adjust the balance between antitumour and anticoagulation effects, facilitating the potent application of TBA in antitumour therapy with reduced side effects. AS1411 (5′-GGT GGT GGT GGT TGT GGT GGT GGT GG-3′) is another G4 aptamer that has shown strong binding affinity to nucleolin (NCL).¹⁵ Since NCL is a multifunctional nonribosomal protein that interacts with both DNA and RNA, the binding of AS1411 to NCL could interfere with a variety of cellular activities.^16–19 NCL is overexpressed in many types of cancerous cells.²⁰ It can bind to AU-rich elements in the 3′-untranslated region of Bcl-2 mRNA, thereby stabilizing and protecting Bcl-2 mRNA from RNase T1 degradation.²¹ NCL also represses the translation of p53 mRNA, which depends on both 5′- and 3′-UTR sequences.²² AS1411 has been reported to induce tumour cell apoptosis by downregulating Bcl-2 by binding with NCL.²³ Another study showed that AS1411 induced cell apoptosis and cycle arrest and inhibited cell viability by upregulating p53 and downregulating Bcl-2 and Akt1 in human glioma cells.²⁴

Although TBA and AS1411 form different G-quadruplex structures, their antitumour activities are both related to the ability to bind to a GRO-binding protein, which has been tentatively identified as NCL.²⁵ In addition, the results of a recent study on AS1411 revealed that the antiproliferative properties of GROs were due to multitargeted effects.²⁶

In our previous work, characteristic chemical modification strategies were applied to alter the affinity of G4 aptamers to their targets, including isonucleoside, 2′-deoxyinosine incorporation and phosphate backbone alkylation.^27–29 Furthermore, we developed DXBA, an outstanding representative of a novel class of nucleoside-based lipids, to solve the problem of nucleic acid drug delivery. DXBA can interact with oligonucleotides via H-bonding and pi-stacking, and the cytidinyl lipid DNCA has been demonstrated to be an effective neutral transfection material for G4 aptamer delivery. AS1411/DNCA nanoparticles showed high antiproliferative activity (>50%) at low concentrations in A549, MCF-7 and A549/TXL cells.³⁰ In addition, modified antisense nucleic acids G3139 and CT102 encapsulated by cytidinyl-lipid/cationic lipid (DNCA/CLD) were able to enter cells, and a high proportion of nuclear colocalization was observed.^31,32

In the present study, we used DNCA to deliver G4 aptamers into the human lung adenocarcinoma A549 cell line and the drug-resistant A549/TXL cell line. At low concentrations (100 nM), AS1411/DNCA and TBA/DNCA both showed excellent antitumour activity against A549/TXL cells, and their nuclear localization signals were detected. hnRNP A1 was identified as a target protein of AS1411 and TBA. More interestingly, we first confirmed that AS1411 can not only target the free hnRNP A1 protein but also target the KRAS promoter/hnRNP A1 complexes, while TBA had almost no significant competitive effect.

Results

G4 aptamer showed increased potency against drug-resistant lung cancer

Lung cancer is a malignant tumour with very high mortality and invariably emerging drug resistance after treatment. Nanomedicines have great potential to combat drug resistance, because of their excellent permeability and retention effects.³³ In this study, a neutral cytidinyl lipid, DNCA, was used to transfect and deliver G4 aptamers (AS1411, TBA) into tumour cells, including multidrug-resistant tumour cells. According to the results of circular dichroism (CD) spectroscopy (Fig. 1A), AS1411 and TBA can form different G4 structures, with characteristic peaks at 260 nm and 290 nm, respectively. The effect of the G4 aptamer (encapsulated by DNCA) on cell viability was tested in the human lung fibroblast line HFL1, the human lung carcinoma cell line A549, the cisplatin-resistant cell line A549/DDP, and the taxol-resistant cell line A549/TXL. The concentrations of aptamers were 100 nM, and the sequence CCT TCC TCT CCT TCC was used as a negative control (NC). The G4 aptamer encapsulated by DNCA clearly showed high antitumour activity, while the naked aptamer displayed almost no activity at the same concentration. And both TBA/DNCA and AS1411/DNCA showed stronger effects on antiproliferation of drug-resistant tumour cells, especially A549/TXL cells, than nondrug-resistant cells (Fig. 1C and S5†). TBA/DNCA and AS1411/DNCA also showed more potency against multidrug-resistant tumours when we investigated their effects on the growth of MCF-7 and MCF-7/ADR cells (Fig. S8†). Besides, all experimental groups had no significant effect on the growth of HFL1 cells (Fig. 1B), indicated the safety of DNCA lipid.

Fig. 1 A. CD spectra of TBA and AS1411. B. Effects of TBA and AS1411 on the growth of HFL1 cells. C. Effects of TBA and AS1411 on the growth of A549, A549/TXL and A549/DDP cells. Cell viability was assayed 48 h after the addition of aptamer (aptamer or NC: 100 nM, DNCA: 7.5 μM) using the CCK-8 assay. A pool of three different sets of experiments (each repeated in triplicate) was performed, and each value was expressed as the mean ± standard deviation.

Annexin V/PI staining revealed that AS1411/DNCA significantly promoted apoptosis (approximately 26% increase in apoptotic cells) in A549/TXL cells, especially early apoptosis (approximately 23% increase in early apoptotic cells) (Fig. 2). In A549 cells, AS1411/DNCA also promoted apoptosis (approximately 12% increase in apoptotic cells), but the effect was greatly decreased. Compared with AS1411/DNCA, TBA/DNCA had a weaker effect against both cell lines. However, TBA/DNCA also showed stronger pro-apoptotic effects in multidrug-resistant cells (the apoptosis rate of A549 cells increased by approximately 7%, and the apoptosis rate of A549/TXL cells increased by approximately 14%).

Fig. 2 Effects of TBA/DNCA and AS1411/DNCA on cell apoptosis. Apoptosis was assayed 24 h after the addition of aptamer by flow cytometry. A pool of three different sets of experiments (each repeated in triplicate) was performed, and each value was expressed as the mean ± standard deviation (aptamer or NC: 100 nM, DNCA: 7.5 μM).

The effects of the G4 aptamer (encapsulated by DNCA) on the cell cycle were also detected. The results (Fig. 3A) showed that both AS1411/DNCA and TBA/DNCA can induce S-phase arrest in A549/TXL cells. AS1411/DNCA also induced S-phase arrest in A549 cells, but the effect was weaker than that in multidrug-resistant cells. TBA/DNCA did not induce S-phase arrest in A549 cells. In summary, two aptamers that can form different G4 structures exhibited increased potencies against multidrug-resistant cells, and their antitumour effects have similarities and differences.

Fig. 3 A. Effects of TBA and AS1411 on the cell cycle. B. Distributions of TBA and AS1411 in the nucleus of A549/TXL cells. The cell cycle was assayed by flow cytometry 24 h after the addition of aptamer, and the cellular distribution was analysed by confocal microscopy 8 h after the addition of FAM-labelled aptamer. A pool of three different sets of experiments (each repeated in triplicate) was performed (aptamer or NC: 100 nM, DNCA: 7.5 μM).

Antitumour mechanism of the G4 aptamer

The cellular uptake of the FAM-labelled G4 aptamer was analysed in the A549 cell line and the A549/TXL cell line by flow cytometry (Fig. S6A†) and confocal microscopy (Fig. S6B†). In both cell lines, the G4 aptamer encapsulated by DNCA had higher cellular uptake than the naked aptamer. For AS1411, the increases were more pronounced. No significant colocalization of the FAM-labelled aptamer and LysoBrite™ NIR was detected, which indicated that the delivery of the DNCA-encapsulated G4 aptamer into cells prevented degradation of the aptamer in lysosomes. On the other hand, comparing the NC-related results showed that not all sequences can be efficiently loaded into the cell by DNCA. It is speculated that DNCA may increase the cellular uptake of the aptamers through the formation of particles based on their G4 structures. More importantly, cellular uptake and inhibitory activity were not strictly positively related, indicating that G4 aptamers have higher antitumour efficacies in drug-resistant cells, which may be related not only to the amount of cellular uptake but also to the intracellular target interaction. The high uptake and low efficacy of TBA against A549 cells further supported this.

We also observed through confocal microscopy that the G4 aptamer entered the nucleus 8 h after being added to the cell culture medium (Fig. 3B), which indicated that the G4 aptamer may also play a regulatory role in the nucleus. To conduct a preliminary exploration of the antitumour mechanism of the G4 aptamer, we conducted a proteomics experiment. After 24 h of drug incubation, cells were collected and digested to extract the proteins, which were then degraded by pancreatin, and the peptide fragments were further labelled by iTRAQ reagents. Through LC-MS analysis, large numbers of peptides, proteins and quantifiable proteins were found. A t-test was used to screen for proteins with significant differences in expression; “significant” is defined as a p value less than 0.05 and an expression difference larger than a 1.2 ratio. Compared with the control group, the TBA/DNCA and AS1411/DNCA groups all displayed noteworthy regulatory effects on protein expression. Comparing the AS1411/DNCA and TBA/DNCA groups showed small differences in regulatory effects on some proteins (Fig. 4).

Fig. 4 Protein expression comparison. A pool of three different sets of iTRAQ-based quantitative proteomics experiments (each repeated in triplicate) was performed (aptamer: 100 nM, DNCA: 7.5 μM) in A549/TXL cells.

To further determine the target and pathway of the G4 aptamer, a BLI experiment was carried out. The peptide-spectrum matching (PSM) value can reflect the relative amount of protein detected by mass spectrometry. As shown in Table 1, the major targets of AS1411 and TBA are hnRNP A1, hnRNP M, NCL and YB-1. These four proteins play important roles in the development of tumours. This result strongly indicated that the intracellular protein targets of AS1411 were not limited to NCL, and the detailed mechanism remains to be elucidated.

Table 1 Peptide-spectrum matching (PSM) value in the BLI experiment

Protein	PSM
	AS1411	TBA
hnRNP A1	21.0 ± 4.00	21.0 ± 5.00
hnRNP M	19.5 ± 5.00	16.5 ± 5.00
Nucleolin	16.0 ± 1.00	9.00 ± 0
YB-1	8.00 ± 5.00	11.0 ± 4.00

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The proteomics results indicated that the proteins regulated by AS1411/DNCA and TBA/DNCA were involved in the KRAS pathway, multiple p53-related pathways and the MYBBP1A protein, which is mainly located in the nucleus. Moreover, the effects of AS1411/DNCA and TBA/DNCA on numerous ribosome-associated proteins and ubiquitination-related proteins were also detected (Tables S1–S4†). According to the literature, high expression of the MYBBP1A protein can lead to defects in pre-rRNA processing in cells,³⁴ and Pol I transcription is inhibited when NCL/rDNA G-quadruplex complexes are disrupted in the nucleolus.³⁵ A gradual reduction in rRNA transcription triggers p53 acetylation and tumour cell apoptosis.³⁶ Moreover, ubiquitination is an important regulatory mechanism of p53,^37,38 thus, all these factors make up a correlative network. In conclusion, both TBA/DNCA and AS1411/DNCA exerted antitumour effects on A549/TXL cells mainly through the KRAS pathway, p53-related biological processes and epigenetic regulation of rRNA expression. We planned to conduct in-depth research on the relevant mechanisms of the KRAS pathway and organize the relevant iTRAQ results, as shown in Fig. 5. Notably, KRAS target is attractive target for antitumour drugs development.³⁹ Activated KRAS protein can activate the rapidly accelerated fibrosarcoma (RAF)-mitogen-activated protein kinase (MEK)-extracellular regulated protein kinases (ERK) signalling pathway, and other multiple signalling pathways.^40,41

Fig. 5 Effector proteins in the antitumour pathway of TBA/DNCA and AS1411/DNCA. A pool of three different sets of iTRAQ-based quantitative proteomics experiments (each repeated in triplicate) was performed (aptamer: 100 nM, DNCA: 7.5 μM) in A549/TXL cells.

hnRNP A1-the newly identified target protein of AS1411 and TBA

To verify the results regarding hnRNP A1 in Table 1, a BLI experiment was performed to measure the affinity between the G4 aptamer and hnRNP A1 protein. The results showed that for TBA, the K_d value was 21.1 nM (Fig. 6A), and for AS1411, the K_d value was 17.5 nM (Fig. 6B). hnRNP A1 can bind to the G4 structure in the promoter region of the intractable oncogene KRAS and open this structure, thereby promoting KRAS expression.^7,42,43 Combined with the results of Fig. 3B and 5, both TBA and AS1411 (encapsulated by DNCA) can enter the cell nucleus, and both may affect the tumour-associated KRAS-RAF1-ERK1/2 (p-ERK1/2) pathway. Therefore, the effects of TBA and AS1411 on KRAS mRNA expression were quantitatively analysed by RT qPCR. The results showed that in A549/TXL cells, KRAS mRNA was reduced by approximately 40% 24 hours after administration of TBA or AS1411 (Fig. 6D), indicating that the two G4 aptamers might competitively bind to hnRNP A1 and block the hnRNP A1/KRAS promoter G4 interaction, leading to KRAS mRNA inhibition. On the other hand, the expression level of KRAS mRNA in A549/TXL cells was approximately 9.4-fold higher than that in A549 cells (Fig. 6C), suggesting a close relationship between KRAS and multidrug resistance in tumour cells. Therefore, the downregulation of KRAS by TBA and AS1411 may be one of the reasons they exhibited selective antitumour effects against multidrug-resistant tumour cells. Western blot experiments also showed that both TBA and AS1411 reduced the expression of the KRAS protein and the downstream protein phosphorylated ERK1/2 (p-ERK1/2) (Fig. 6E).

Fig. 6 A. Determination of the binding affinity of TBA for the hnRNP A1 protein. B. Determination of the binding affinity of AS1411 for the hnRNP A1 protein. C. Quantitative analysis of KRAS mRNA expression in A549 and A549/TXL cells. D. Effects of TBA and AS1411 on KRAS mRNA expression in A549/TXL cells (****P < 0.0001). E. Effects of TBA and AS1411 on KRAS, ERK1/2 and phospho-ERK1/2 protein expression in A549/TXL cells (aptamer: 100 nM, DNCA: 7.5 μM).

AS1411 can target KRAS promoter/hnRNP A1 complexes

The binding of hnRNP A1 to KRAS promoter G4 has been reported in several studies. Susanna Cogoi and co-workers investigated the recruitment of hnRNP A1 and MAZ to the promoter G4 region (32R). They performed chromatin immunoprecipitation (ChIP) and qPCR experiment with Panc-1 cells treated with H₂O₂ or without treatment. The recruitment and binding of hnRNP A1 and MAZ to the KRAS promoter were detected and the results show that the H₂O₂ treatment increases the recruitment of hnRNP A1 and MAZ, because of a cellular increase of 8-oxoG.⁴⁴ In another study, the regulation effect of integrin-linked kinase (ILK) in KRAS expression was confirmed. The activity of the KRAS promoter was increased by the ectopic expression of ILK in the dual-luciferase reporter assay, and further investigation showed that ILK regulated KRAS expression through hnRNPA1-mediated transcriptional activation.⁴⁵

Thus, BLI competition binding experiments revealed the molecular mechanism by which AS1411 regulates KRAS expression. The KRAS promoter was captured on the biosensor and then dipped into wells containing hnRNP A1 alone or with aptamer, which showed that AS1411 partly blocks hnRNP A1 from binding to the KRAS promoter.⁴⁶ After binding to hnRNP A1 alone, the biosensor with the KRAS promoter–hnRNPA1 complex was dipped into wells containing aptamer or buffer. The results showed that AS1411 can not only bind to free hnRNP A1 but also accelerate hnRNP A1 dissociation from the KRAS promoter, which confirmed that AS1411 could recognize the same epitope as the KRAS promoter. In contrast, TBA had no significant competitive effect in vitro (Fig. 7A).

Fig. 7 A. Results of the competitive binding experiment. BLI assays were conducted using an Octet RED 96 system with SA sensors to study the interaction between hnRNP A1, the KRAS promoter and the G4 aptamer (AS1411, TBA). B. The binding pattern of the KRAS promoter/hnRNP A1 (light red ribbon) and AS1411/hnRNP A1 (cyan ribbon) complexes predicted by MD simulation. C. Local structures of key binding sites of the KRAS promoter/hnRNP A1 complex. D. Local structures of key binding sites of the AS1411/hnRNP A1 complex. E. The binding pattern of TBA/hnRNP A1 (light red ribbon) complexes predicted by MD simulation. F. Local structures of key binding sites of the TBA/hnRNP A1 complex. The purple parts of the ribbons of the nucleic acids represent loops. The potassium ions fixing the G4 structure are represented by orange balls. The surface of the protein is shown in blue (hydrophilic parts) and red (lipophilic parts). Hydrogen bonds are shown by green dotted lines, and π–π and H–π interactions are shown by red dotted lines.

Molecular docking and molecular dynamics (MD) simulations were performed to explore the binding patterns of several representative G4 aptamer/hnRNP A1 complexes. The RNA binding motif of hnRNP A1 (PDB ID: 2UP1) with two binding domains was used as a receptor. The results showed that all G4 structures fixed by potassium ion(s) remained stable during binding and that AS1411 (dimer) and the KRAS promoter bound to the same domain (Fig. 7B–D), while TBA tended to bind to another domain (Fig. 7E and F). This supported the conclusion that AS1411 could recognize the same epitope (interaction site) as the KRAS promoter.

In the binding patterns of AS1411/hnRNP A1 and KRAS promoter/hnRNP A1, the binding pocket formed by several arginines and phenylalanines played a significant role. The loops of AS1411 (T12-T13-G14-T15) and the KRAS promoter (A14-A15-T16-A17) formed close contacts with this binding pocket, and hydrogen bonds (formed with Arg48) were the main interactions. The poses of the loops (Fig. 7B) might determine the competitive relationship between AS1411 and the KRAS promoter when binding to this domain of hnRNP A1.

In the binding patterns of TBA/hnRNP A1, a similar binding pocket was shown in the other domain. Notably, the two TT loops in TBA, which were proven to be interaction sites binding to thrombin (PDB ID: 4DII), also formed a degree of close contacts with hnRNP A1, guaranteeing the interaction between TBA and hnRNP A1.

Discussion

Exogenous G-quadruplexes have enormous application prospects. We focused on two G4 aptamers, TBA and AS1411, that have shown excellent antitumour activity. In previous studies, poor cellular permeability limited their potential for widespread application. Although considerable research has focused on improving their affinity for targets through chemical modification, the limitations of dosage and administration have not yet been completely solved. Here, a suitable carrier, DNCA, was used to deliver G4 aptamers into tumour cells. In this effective delivery strategy, G4 aptamers exhibited high efficacies at a low concentration (reduced from ∼10 μM to 100 nM). More interestingly, both AS1411 and TBA showed selective antitumour activities. Thus, G4 aptamer/DNCA can be developed into a complex drug system that specifically targets drug-resistant tumours. The combination of the G4 aptamer with a specific carrier to specifically combat drug-resistant tumours in this study has provided new ideas for research in related fields.

In mechanistic research, we found that the expression of KRAS mRNA in the drug-resistant tumour cell line A549/TXL was higher (9.4-fold) than that in the A549 cell line and that the two G4 aptamers lowered the KRAS mRNA and KRAS protein levels in the A549/TXL cells. This was a possible reason why the two G4 aptamers showed more potency against drug-resistant tumour cells. hnRNP A1 was newly identified as a target protein of the two aptamers. The results of competition experiments showed that AS1411 can target not only free hnRNP A1 protein but also target KRAS promoter/hnRNP A1 complexes, while TBA had no significant competitive effect.

In conclusion, AS1411 targets the protein (hnRNP A1)–nucleic acid (KRAS promoter G4) complex and competitively binds to hnRNP A1. The KRAS promoter G4 structure cannot be unfolded by hnRNPA1, leading to KRAS transcription inhibition and tumour cell death, especially in drug-resistant cells. Although TBA cannot compete with the KRAS promoter to bind hnRNP A1, the BLI experiment clearly showed that TBA and AS1411 have similar effects on several key proteins in tumour cells, especially hnRNP A1, thus strongly suggesting that AS1411 can directly interact with hnRNP A1, while TBA interferes with the biological function of hnRNP A1 indirectly by mechanisms that will need to be revealed in further research.

In addition, based on the results of iTRAQ-based quantitative proteomics analysis and BLI experiments, we plan to continue to explore the effects of G4 aptamers on nuclear/rDNA G-quadruplex complexes and epigenetic-related processes, as well as examining tumour inhibition at the animal level.⁴⁷

Materials and methods

DNA aptamer

The sequences were synthesized on an ABI 394 synthesizer and purified with HPLC (linear gradient using 5–35% acetonitrile–100 mM TEAB 100 mM in 20 min, XBridge™ OST C18 2.5 μm 10 × 50 mm column, 60 °C, 1.5 mL min⁻¹, 260 nm, Fig. S1†). ESI-TOF was used for characterization (Fig. S2–S4†).

Circular dichroism spectroscopy

Circular dichroism spectra of modified TBAs and AS1411s were obtained with a Jasco J610 spectrometer (Japan) using 0.5 mL quartz cuvettes with a 2 mm path length. The concentrations of all oligonucleotides were 7 μM. The oligonucleotides were dissolved in 1× phosphate buffered saline buffer (138 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.76 mM KH₂PO₄, pH 7.2–7.4), annealed on a PCR instrument (95 °C, 10 min, then reduced the temperature by 5 °C per 5 min until 25 °C). The measurement was performed at 25 °C, and the wavelength ranged from 220 nm to 320 nm. Data were smoothed using the system software.

Cell line and culture conditions

A549, A549/TXL and A549/DDP cell lines (Keygen Biotech, China) were cultured in Roswell Park Memorial Institute-1640 medium with L-glutamine (RPMI-1640, Corning) supplemented with 10% foetal bovine serum (Gibco) under a humidified atmosphere of 5% CO₂ at 37 °C.

Cell viability assay

Cells were seeded into 96-well plates at a density of 3 × 10³ cells per well and grown for 24 h. Then, 100 nM aptamer and 7.5 μM DNCA mixtures or GenOpti (M&C Gene Technology) as a control were added to the culture medium. After 48 h, cell viability was assayed using the Cell Counting Kit-8 (CCK-8) (Dojindo Laboratories): after the cells were incubated with CCK-8 solution for 1.5 h, optical density was measured at 450 nm using a microplate reader (FlexStation 3).

Cellular uptake study

Cells were seeded into 6-well plates at a density of 2 × 10⁵ cells per well and grown for 24 h. FAM-labelled aptamer (100 nM) and DNCA mixtures (7.5 μM) or GenOpti (M&C Gene Technology) as a control were then added to the culture medium. After 4 h, the cells were harvested and washed three times with PBS. Then, cellular uptake was determined by flow cytometry.

Confocal microscopy

Cells were grown on confocal observation dishes for 24 h. FAM-labelled aptamer (100 nM) and 7.5 μM DNCA mixtures or GenOpti (M&C Gene Technology) as a control were then added to the culture medium. After 4 h, the culture medium was removed, and the cells were washed twice with PBS. Then, the cells were stained with Hoechst 33342 (Solarbio) and LysoBrite™ NIR (AAT Bioquest) for 30 min and observed under an A1Rsi confocal microscope (Nikon Instruments Inc.). Confocal images were obtained using NIS-Elements and Bitplane Imaris software (Nikon Instruments Inc.).

Apoptosis assay

Cell apoptosis was quantified using an Annexin V-FITC/PI apoptosis detection kit (BD Pharmingen). After treatment, cells seeded into 6-well plates were harvested, washed twice with cold PBS and resuspended in 100 μL of 1× binding buffer. Next, 5 μL of Annexin V-FITC and 5 μL of PI were added to each sample, and the cells were incubated at room temperature for 20 min in the dark. The stained samples were then analysed by a FACSCalibur™ cytometer (BD Biosciences).

Flow cytometry analysis of the cell cycle

Cells were plated into 6-well plates at a density of 2 × 10⁵ cells per well and grown for 24 h. After treatment, cells were harvested by trypsinization, washed with cold PBS twice, and fixed in cold 70% ethanol for 24 h at 4 °C. Then, the cells were treated with RNase A (Solarbio), stained with propidium iodide (Solarbio) and analysed using a FACSCalibur™ cytometer (BD Biosciences). The percentage of cells in G1/G0, S and G2/M phases was determined using the Modfit program (Verity Software House).

Cellular proteomics study

After treatment, the cells were harvested by trypsinization and washed twice with cold PBS. Then, 400 μL of lysis buffer (7 M carbamide, 2 M thiocarbamide, 0.1% PMSF, 65 mM DTT) was added per 10⁶ cells. The cells were broken by ice bath ultrasonication (70–75 W, 5 s on, 10 s off, repeated 3–5 times) and then incubated on ice for 40 min before being centrifuged at 14 000 rpm for 30 min at 4 °C. The supernatants were collected, and total protein concentrations were determined using the Bradford protein assay. Protein digestion was conducted according to the FASP (filter aided sample preparation) procedure. In brief, 200 μg of protein was solubilized in 10 μL of reducing reagent at 37 °C for 1 h. Then, 2 μL of cysteine-blocking reagent was added, and the mixture was incubated at room temperature for 30 min before being centrifuged at 12 000 rpm for 20 min. Each filter was washed three times with 300 μL (100 μL per wash) of dissolution buffer (Applied Biosystems) by centrifugation at 12 000 rpm for 20 min. Proteins were then digested in solution with trypsin (Promega) according to a protein/trypsin ratio of 50 : 1 at 37 °C overnight. The digested peptides were labelled with iTRAQ reagents (Applied Biosystems). The labelled peptide fragments from each sample were reconstituted with 100 μL of buffer A (98% ddH₂O, 2% acetonitrile, pH 10) before being centrifuged at 14 000 rpm for 10 min and then eluted with gradient buffer B (98% acetonitrile, 2% ddH₂O, pH 10) using a RIGOL L-3000 high-performance liquid chromatography system (Beijing RIGOL Technology Co., Ltd.) with an RP analytical column (Durashell-C18, 4.6 mm × 250 mm, 5 μm, 100 Å) at a flow rate of 0.7 mL min⁻¹. Fractions were collected, and all samples were redissolved in 2% methyl alcohol and 0.1% formic acid followed by centrifugation at 13 000 rpm for 10 minutes. LC-MS/MS was carried out using an Easy-nLC nanoflow HPLC system connected to a Q Exactive™ HF mass spectrometer (Thermo Fisher Scientific). iTRAQ labelling, LC-MS/MS analysis and protein identification were accomplished in cooperation with Haiteng Bio, Beijing.

Target fishing experiment

BLI was performed to investigate the targets of AS1411 and TBA (Sangon). Nucleoproteins of A549/TXL cells were obtained using a Nuclear Protein Extraction kit (Solarbio). Biotin aptamers were loaded onto super streptavidin (SSA) biosensors, and then the biosensors were incubated with nucleoproteins from A549/TXL cells for 300 s. The proteins bound to biosensors were washed into 0.1% formic acid solution and collected for MS analysis.

Affinity detection

BLI was performed to detect the affinity between the aptamer (Sangon) and hnRNP A1 protein (Abcam). Biotin aptamers were loaded onto super streptavidin (SSA) biosensors, and then the biosensors were incubated with hnRNP A1 protein solution at different concentrations (600 nM, 300 nM, 150 nM, 75 nM, 37.5 nM, 18.8 nM, 9.38 nM, 0 nM). The association and dissociation between the aptamer and hnRNP A1 were detected. K_d values were acquired by using ForteBio Data Analysis software.

Real-time PCR assay

Cells were seeded into 6-well plates at a density of 2 × 10⁵ cells per well and grown for 24 h. Then, 100 nM aptamer and 7.5 μM DNCA mixtures or GenOpti (M&C Gene Technology) as a control were added to the culture medium. After 24 h, the cells were harvested, and RNA was extracted from the cells using TRIzol reagent (Life Technologies) according to the standard extraction protocol. One microgram of total RNA was subjected to reverse transcription using the Reverse Transcription System (Promega). Then, quantitative real-time PCR was carried out using the Mx3005P QPCR System (Agilent) with SYBR Green QPCR Master Mix (Promega). The primers (5′-3′) used in this study are listed in Table S5.† The real-time PCR followed a three-step amplification protocol (95 °C, 30 s; 61 °C, 60 s; 72 °C, 60 s; 35 cycles) followed by a melting curve segment (95 °C, 60 s; 55 °C, 30 s; 95 °C, 30 s; 1 cycle). Relative gene expression was normalized to that of GAPDH and calculated by using the 2^−ΔΔCt (cycle threshold) method.

Western blot

A549/TXL cells were collected 24 h after aptamer (100 nM) transfection by DNCA (7.5 μM). Cells were lysed in 50 μL of lysis buffer (M&C Gene Technology) for 1 h on ice, and the lysis reaction was mixed by pipetting every 15 min. The lysates were clarified by centrifugation for 10 min at 16 000 × g, and the protein concentration was determined by a BCA kit (Solarbio). Total protein was separated on 30% Bis-Tris-polyacrylamide gels and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore) at 400 mA for 2 h. After being blocked using 5% skim milk for 2 h, the membrane was first incubated with anti-Ras (or ERK1/2 or phospho-ERK1-T202/Y204 + ERK2-T185/Y187 or β-tubulin) polyclonal antibody (Solarbio) in a primary antibody dilution buffer at 4 °C overnight. Then, the cells were washed and incubated using a goat anti-mouse monoclonal antibody (Abcam) in 5% skim milk for 2 h. Finally, the protein products were analysed using Image Lab software (ChemiDoc XRS System, Bio-Rad).

Competitive binding experiment

BLI assays were conducted using an Octet RED 96 System with SA sensors to study the interaction between hnRNP A1, the KRAS promoter and the G4 aptamer. A total of 25 μM 5′-biotin-TEG-AGGGCGGTGTGGGAAGAGGGAAGAGGGGGAGGCAG (KRAS promoter obtained as an HPLC-purified sample from Sangon Biotech) and G4 aptamer were refolded in PBS (pH 7.4) by heating at 95 °C in a heater block for ten minutes and then cooled slowly to room temperature. The G4 aptamer was diluted to 100 nM, and hnRNP A1 was diluted to 200 nM in BLI buffer (20 mM phosphate buffer, 8 mM KCl, 137 mM NaCl, 0.05% surfactant P20) to serve as an analyte.

Molecular docking and molecular dynamics (MD) simulation

Molecular docking was performed using the ZDOCK server⁴⁸ (https://zdock.umassmed.edu/). From the top 10 predictions provided by the ZDOCK server, we chose the poses with a clear majority to be the initial conformation of the MD simulation. The structures of proteins and nucleic acids were provided by the PDB database (https://www.rcsb.org/), including hnRNP A1 (PDB ID: 2UP1),⁴⁹ TBA (PDB ID: 4DII),⁵⁰ and the KRAS promoter (PDB ID: 6T51).⁵¹ The structures of AS1411 were built with the software Schrödinger (https://www.schrodinger.com/).

MD simulation was performed with the software AMBER 18 (ref. 52–55) in the Ubuntu 16.04.7 LTS system. The simulation process included solvation, heat, equilibration and production. The length of the production process was 200 ns, and the process was operated at 300 K. All structures achieved equilibration after these processes, and we regarded the results as stable conformations.

Statistical analysis

GraphPad Prism 8.0, Origin 2022 software was used for statistical analyses of all data. And the p value was obtained by one way ANOVA to determine the significance.

Data and code availability

The data supporting the findings of the present study are available from the corresponding author upon reasonable request.

Author contributions

Z. J. Yang obtained funding, conceptualized the study and was responsible for the project. Y. J. Zhu and X. Li designed and performed the experiment and analyzed the data. Q. Zhang, X. T. Yang, X. D. Sun, Y. Pan, X. Yuan, Y. Ma and B. Xu provided essential experimental support. Y. J. Zhu wrote the manuscript with assistance from additional co-authors.

Conflicts of interest

The authors declare no competing interests.

Acknowledgements

This work was supported by the Ministry of Science and Technology of China (Grant No. 2012CB720604, 2017ZX09303013) and the National Natural Science Foundation of China (Grant No. 21778006).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3md00752a

‡ These authors contributed equally to this paper.

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