Characterization of A Bifunctional Synthetic RNA Aptamer and A Truncated Form for Ability to Inhibit Growth of Non-Small Cell Lung Cancer

An in vitro-transcribed RNA aptamer (trans-RA16) that targets non-small cell lung cancer (NSCLC) was previously identified through in vivo SELEX. Trans-RA16 can specifically target and inhibit human NCI-H460 cells in vitro and xenograft tumors in vivo. Here, in a follow-up study, we obtained a chemically-synthesized version of this RNA aptamer (syn-RA16) and a truncated form, and compared them to trans-RA16 for abilities to target and inhibit NCI-H460 cells. The syn-RA16, preferred for drug development, was by design to differ from trans-RA16 in the extents of RNA modifications by biotin, which may affect RA16’s anti-tumor effects. We observed aptamer binding to NCI-H460 cells with KD values of 24.75 ± 2.28 nM and 12.14 ± 1.46 nM for syn-RA16 and trans-RA16, respectively. Similar to trans-RA16, syn-RA16 was capable of inhibiting NCI-H460 cell viability in a dose-dependent manner. IC50 values were 118.4 nM (n = 4) for syn-RA16 and 105.7 nM (n = 4) for trans-RA16. Further studies using syn-RA16 demonstrated its internalization into NCI-H460 cells and inhibition of NCI-H460 cell growth. Moreover, in vivo imaging demonstrated the gradual accumulation of both syn-RA16 and trans-RA16 at the grafted tumor site, and qRT-PCR showed high retention of syn-RA16 in tumor tissues. In addition, a truncated fragment of trans-RA16 (S3) was identified, which exhibited binding affinity for NCI-H460 cells with a KD value of 63.20 ± 0.91 nM and inhibited NCI-H460 cell growth by 39.32 ± 3.25% at 150 nM. These features of the syn-RA16 and S3 aptamers should facilitate the development of a novel diagnostic or treatment approach for NSCLC in clinical settings.

their targets with specificity and high affinity 12 . Typically, specific aptamers are generated by a process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX) 13,14 . Since the invention of SELEX in the 1990s, specific aptamers for various targets have been identified [15][16][17] .
A previous study by our group demonstrated the potential of a NSCLC-specific RNA aptamer selected via in vivo SELEX 18 . The aptamer, named RA16, was capable of binding to and inhibiting NSCLC human large cell lung cancer cell line NCI-H460 cells in vitro and in vivo, which may be applied to tumor imaging technique and targeted therapies. A major advantage of RNA aptamers is that they can be chemically synthesized for use in diagnosis, treatment and biomarker discovery. Therefore, the binding and inhibitory activity of the synthesized RA16 (syn-RA16), as well as the potential mechanisms should be further investigated. Furthermore, a smaller aptamer size could facilitate large-scale chemical synthesis and would be beneficial for clinical applications.
Here, we conducted a sequential study of the syn-RA16 and truncated aptamers specifically targeted and directly inhibited towards NCI-H460 cells in vitro and in vivo. We also demonstrated the potential tentative mechanism for syn-RA16 internalization and intracellular signaling mechanism.

Results
Specificity and Affinity of syn-RA16 in vitro. We previously reported that the NSCLC-specific RA16 selected via in vivo SELEX could bind to NSCLC NCI-H460 cells in vitro. To further confirm the specificity of the selected syn-RA16 aptamer, its binding activity was evaluated and compared with that of trans-RA16 in vitro. The syn-RA16 or biotin-labeled trans-RA16 aptamer was incubated with NCI-H460 cells, NSCLC (human lung adenocarcinoma cell line SPC-A1 cells), or control cells including human embryonic kidney-293T (HEK-293T) cells, human cervical carcinoma cell line (HeLa cells), and human normal lung cell line (BEAS-2B cells). Scrambled RNA (SCAP) served as the negative control. Fluorescence was detected using streptavidin-Alexa Fluor 488, and the cells were imaged under a microscope. Fluorescent binding was observed for NCI-H460 cells, but not SPC-A1, HEK-293T, HeLa, and BEAS-2B cells (Fig. 1A,B), suggesting that syn-RA16, similar to trans-RA16, demonstrated specific binding to NCI-H460 cells in vitro.
To assess the binding affinity of syn-RA16 and compare it with trans-RA16, flow cytometry was performed. As shown in Fig. 1C, when NCI-H460 cells were incubated with biotin-labeled RNA molecules, syn-RA16, similar to trans-RA16, bound to most of the cells and exhibited a clear fluorescence shift detected by streptavidin-PE, thus indicating the specific binding between syn-RA16 and NCI-H460 cells. There was no noticeable fluorescence shift for the negative control (Fig. 1C). We then determined the binding affinity of syn-RA16 and trans-RA16 for NCI-H460 cells by measuring the equilibrium dissociation constant (K D ). Analysis of the fitted lines revealed K D values of 24.75 ± 2.28 nM and 12.14 ± 1.46 nM for syn-RA16 and trans-RA16, respectively (Fig. 1D), demonstrating the high affinity of the aptamers for NCI-H460 cells.
Specific binding mediated internalization of aptamer RA16. As reported, most cell-binding aptamers rely on cell membrane biomarkers for recognition and internalization through endocytosis [19][20][21] . To further investigate the time course of RA16 and potential mechanism for aptamer internalized, we performed time course study to monitor aptamer syn-RA16 internalization with different incubation time points.
As shown in Fig. 2A, syn-RA16 gradually entered NCI-H460 cells, and binding of syn-RA16-NCI-H460 cells was detectable after 1 h incubation. When the incubation time was increased to 2 h, RA16 gradually entered the cell and signaled at the most in cytoplasm. Colocalization of aptamer RA16 and lysotracker revealed that the most of the aptamer entered cells through endocytosis (Fig. 2B). After 4 h incubation, the syn-RA16 further accumulated in the cytoplasm.
To further quantify time-depending internalization of RA16 to NCI-H460 cells, NCI-H460 cells were incubated with syn-RA16 or SCAP in serum containing medium for different incubation time. The relative aptamer recovery levels were further detected by quantitative RT-PCR. The internalized aptamer kinetics were shown in Fig. 2C. Consistent with the imaging data, SCAP showed low internalization in NCI-H460 cells. On the contrast, syn-RA16 demonstrated a significantly strong internalization. As time increasing, syn-RA16 gradually internalized and reached at the maximum after 4 h incubation.
The results indicated that aptamer RA16 entered NCI-H460 cells through receptor-mediated endocytosis. The specific binding triggered the aptamer internalized, migrated, and finally accumulated in the cytoplasm. The possible mechanism of aptamer binding and signaling courses is shown in Fig. 2D.

inhibition of cell growth in vitro.
Trans-RA16 has been demonstrated previously to inhibit NCI-H460 cell growth 18 . Therefore, we performed cell viability assays to assess the inhibitory activity of syn-RA16. As shown in Fig. 3A, the inhibition rates of NCI-H460 cell growth at various concentrations by syn-RA16 and trans-RA16 were almost similar. Notably, syn-RA16 suppressed NCI-H460 cells by 84.5%, whereas trans-RA16 suppressed the cells by 86.8% at 300 nM. NCI-H460 cells treated with both trans-RA16 and syn-RA16 exhibited an apoptotic phenotype (Fig. 3B). Furthermore, analysis of the inhibition rate with a concentration series of RA16 revealed IC 50 values of 118.4 nM and 105.7 nM for syn-RA16 and trans-RA16, respectively (Fig. 3C). Interestingly, both syn-RA16 and trans-RA16 did not have an inhibitory effect on HeLa cells at 600 nM (Fig. 3D).

Tumor-targeting efficacy in vivo.
After evaluating the specificity and inhibitory activity of syn-RA16 in vitro, we further performed an in vivo tumor imaging assay to investigate the targeting activity of syn-RA16 in vivo. Cy5.5-labeled syn-RA16 or trans-RA16 was injected into NCI-H460 tumor-bearing mice to track the movements of the specific RA16 molecules in vivo. SCAP was used as the control. At 0.5 h, syn-RA16 and trans-RA16 reached the tumor site with a weak fluorescence signal. As time increased, RA16 aptamers gradually accumulated at the tumor site with a strong fluorescence signal at 2 h. Notably, no fluorescence signal was observed in the tumors of mice injected with SCAP (Fig. 4A). Subsequently, we extracted the tumors for imaging; fluorescence signals  Tentative mechanism for RA16 aptamer internalization. (A) Time course for aptamer syn-RA16 entering NCI-H460 cells. After NCI-H460 cells were incubated with syn-RA16 for 0.5, 1, 2, and 4 hours, fluorescence signal of syn-RA16/SCAP was then detected using streptavidin-Alexa Fluor 488 (Scale bar = 35 μm). (B) Representative confocal microscopy images of NCI-H460 cells show significant colocalization between syn-RA16 and LysoTracker (endosome/lysosomes). Alexa-488 staining represents the aptamer, and merged staining represents aptamer(green), endosome/lysosomes(red) and nucleus(blue) localization (Scale bar = 10 μm). (C) Quantification of aptamer syn-RA16 entering NCI-H460 cells. After NCI-H460 cells were incubated with syn-RA16/SCAP for series time points, relative RNA levels in cells were analyzed by qRT-PCR (normalized to GAPDH). All data represent the mean ± SD, n = 4. (D) Mechanism of aptamer-mediated cell binding and intracellular signaling courses. The RA16 specifically bound the aptamer cell biomarkers and internalized in the cells. The aptamer finally accumulated in the cytoplasm, resulting in intracellular signaling pathway. (2019) 9:18836 | https://doi.org/10.1038/s41598-019-55280-x www.nature.com/scientificreports www.nature.com/scientificreports/ were much higher in mice injected with syn-RA16 or trans-RA16 than in those injected with SCAP ( Fig. 4B), demonstrating the specific target binding of syn-RA16 in vivo.
An in vivo trap assay with quantitative reverse transcription polymerase chain reaction (qRT-PCR) was also performed to evaluate the distribution of syn-RA16 in vivo. NCI-H460 tumor-bearing mice were injected with syn-RA16, trans-RA16, or SCAP. Total RNA was then collected from various tissues for RNA quantification (normalized to mouse 18S RNA). The graph of RNA distribution is shown in Fig. 4C (n = 4). The level of syn-RA16 was significantly higher (50-to 1000-fold) in tumor tissues than in other tissues such as liver, kidney, heart, and lung tissues. Similar results were observed for trans-RA16. Moreover, the level of RNA molecules in the tumor tissues was significantly different between the RA16 groups and SCAP group. As shown in Fig. 4C, trapped RA16 was 100-fold higher than trapped SCAP at the tumor site, indicating that the entrapment of syn-RA16 in tumor tissues may be attributed to its specific binding in vivo. Overall, the results demonstrated the tumor-specific targeting activity of syn-RA16 and trans-RA16 both in vitro and in vivo.
Cell binding and inhibitory activity of a truncated aptamer. In order to minimize the functional motif and facilitate large-scale chemical synthesis, RA16 aptamers were truncated into three smaller parts (S1 containing 40 random nucleotides, S2 containing 40 nucleotides with the 3′-end, and S3 containing the 5′-end with 40 random nucleotides). The potential secondary structures of RA16 and three smaller fragments were predicted by Mfold software (http://unafold.rna.albany.edu/?q=mfold), as shown in Fig. 5A 22 .
Binding activity was assessed by flow cytometry. As shown in Figs. 5B and S1, S2, and SCAP (negative control) were unable to bind to the target cells. However, S3, similar to RA16, exhibited binding affinity for NCI-H460 cells. The binding affinity (K D ) was 63.20 ± 0.91 nM (Fig. 5C). www.nature.com/scientificreports www.nature.com/scientificreports/ In addition, the inhibitory activity of S3 was further determined. As shown in Fig. 5D,E, similar to RA16, NCI-H460 cells treated with S3 exhibited an apoptotic phenotype. S3 inhibited H460 cell growth by 39.32 ± 3.25% at 150 nM, while RA16 suppressed the cells by 61.79 ± 3.27%. These results indicated that S3 containing the 5′-end with 40 random nucleotides of RA16 retained cell-binding and inhibitory activity.  www.nature.com/scientificreports www.nature.com/scientificreports/ There have been various efforts to develop novel targeted therapeutics and overcome the drawbacks of chemotherapy. In comparison with chemotherapy, small molecules such as epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been reported to play a major role in enhancing the survival rate of patients with NSCLC and decreasing toxicity 23 . However, studies have reported that EGFR TKIs did not affect patients harboring wild-type EGFR 6,24 . Furthermore, resistance can invariably occur 23,24 . Monoclonal antibodies such as cetuximab and bevacizumab may be effective in attenuating the progression of lung cancer 7,8,10 . However, they face similar resistance problems 23 . In comparison with tumor-targeting monoclonal antibodies, aptamers have several advantages such as (i) production via chemical synthesis, (ii) no or low immunogenicity, (iii) a smaller molecular size, (iv) efficient biological compartments penetration, and (v) ease of conjugation to various nanomaterials [25][26][27][28] . Previous studies have revealed that specific cancer aptamers are useful for in vitro tumor diagnosis, in vivo tumor imaging technique [29][30][31][32] , and targeted tumor therapy 2,20,33-35 .
Owing to their smaller size, specific binding, and tissue-penetration activity, RNA aptamers are considered as ideal agents for cancer diagnosis and cancer-targeted therapy. Aptamers specific for cancer-related proteins including vascular endothelial growth factor (VEGF), EGFR, mucin 1 (MUC1), and p53 have been identified 15,31,32,36 . Previous studies on targeted chemotherapeutic delivery and tumor imaging have demonstrated the potential of aptamers for targeted treatment and cancer diagnosis 29,30,33,[37][38][39][40] . Recently, an NSCLC-specific RNA aptamer was selected via in vivo SELEX 18 . Binding activity of RA16 to NSCLC cell line (NCI-H1299, SPC-A1, and NCI-H1650 cells), as well as non-NSCLC (HeLa and 293 T cells) were detected respectively, which demonstrated high specificity and affinity towards specific NSCLC tumors. A major advantage of aptamers is the ease of chemical synthesis. Giving synthetic RNA aptamers have a more uniform and highly purified consistent stable structure, the syn-RA16 could easily be adopted for large-scale and cost-efficient production in clinical application. In addition, the syn-RA16 would be beneficial for further modifications such as incorporation of 2′-F dCTP/UTP and 5′-PEGylation, as well chemical adducting and manufacturing 18 . Obviously, the advantages of synthesized aptamers would be more feasible for applications of the clinic.
In this study, we evaluated the specific target binding and direct inhibitory activity of syn-RA16. As we tested and determined the binding affinity in the preliminary study, most of the non-NSCLC cell line showed no or little binding towards RA16, even at high concentration of syn-RA16 at 600 nM. It is our understanding that it's impossible to determine the dissociation constant in lung normal cell lines and in non-NSCLC cell lines. We only determine the dissociation constant in NSCLC H460 cells. Although nucleotide sequences of syn-RA16 and transcribed RA16 are basically the same, syn-RA16 was produced by Dharmacon (GE Healthcare, Lafayette, CO), and trans-RA16 was transcribed from a DNA template in vitro. The main difference between syn-RA16 and trans-RA16 are their labeling status and purity. The affinity of syn-RA16 was slightly lower than that of trans-RA16 as demonstrated by K D determination assay. This result may be attributed to the presence of only one biotin-labeled site in syn-RA16. On the other hand, additional biotin-labeled sites could be incorporated during the in vitro transcription process, resulting in a more sensitive fluorescence signal produced by trans-RA16. However, inhibitory activity was almost similar based on IC 50 values for both syn-RA16 and trans-RA16 (118.4 nM vs. 105.7 nM). We also assessed the specific targeting of syn-RA16 by in vivo tumor imaging and qRT-PCR. Both syn-RA16 and trans-RA16 showed high retention in NCI-H460 tumor tissues in vivo. In fact, a more uniform flow binding profile with syn-RA16 was observed (Fig. 1C), indicating that the preparation of syn-RA16 was more purified than the trans-RA16. This result is consistent with a more tumor and lung bindings for syn-RA16 because with the same molar unit preparations of syn-RA16 and trans-RA16, the more active syn-RA16 aptamers were binding to tumor or lung tissues of NSCLC (Fig. 4C).
In addition, we further investigated the potential mechanism for aptamer binding and growth-inhibiting effects. Based on the time course and cell cycle analysis studies, it is our tentative hypothesis that aptamer RA16 firstly bound to the cell and triggered internalization, followed by further migration and accumulation in the cytoplasm. The internalized aptamer RA16 may regulate some intracellular pathways of NCI-H460 cells, such as, interfering the processes of the protein transcribing or translating in the cell cytoplasm. As a result, these effects may inhibit NCI-H460 cell growth. The tentative mechanism of the aptamer in cell binding and inhibition was proposed as shown in Fig. 2D. Moreover, our preliminary data (unpublished) showed that the target of RA16 is most likely a protein-related component.
On the other hand, a truncated fragment of RA16 (S3) was found to exhibit cell binding and inhibitory activity. Notably, more than three structures for S1 were predicted and only one possible structure for RA16 and S3 was predicted (as shown in Fig. 5A). S3 retained the secondary structure of RA16 at the 5′ end, and the other two truncated fragments (potentially folded into other structures) did not bind to target cells, indicating that the secondary/tertiary structure plays a major role in aptamer activity. The 5′-end structure of RA16 could be critical for specific binding and intracellular signaling. This region may induce aptamer internalization and lead to intracellular signaling for cell growth inhibition. However, the affinity (K D ) and inhibitory effect of S3 were slightly lower than that of the full-length aptamer (63.20 ± 0.91 nM vs. 12.14 ± 1.46 nM; 39.32 ± 3.25% vs. 61.79 ± 3.27%) which is largely consistent with the truncation process, indicating that the 3′-end of the aptamer may also contribute to structure folding and target binding 41,42 .
In conclusion, we conducted a sequential study of the anti-NSCLC aptamer RA16, which can be chemically synthesized. In this study, syn-RA16 demonstrated specificity and high affinity for NSCLC NCI-H460 cells in vitro and in vivo. Notably, we demonstrated the tentative mechanism for syn-RA16 binding and intracellular signaling. In addition, the truncated aptamer (S3) exhibited cell binding and inhibitory activity, indicating that this aptamer could be further truncated and modified. The syn-RA16 and truncated aptamer could contribute to the identification of potential targets and elucidation of molecular mechanisms, which would be beneficial for future applications of the aptamer as a drug or diagnostic reagent for NSCLC. www.nature.com/scientificreports www.nature.com/scientificreports/ 488 according to the manufacturer's protocol. The nucleus was stained with Hoechst 33342 at 37 °C for 5 min and washed with DPBS twice. The cells were then mounted and imaged under a confocal microscope (Olympus, Tokyo, Japan).
Time course study with qRT-PCR. NCI-H460 cells were seeded in 24-well plates at 1 × 10 5 cells per well overnight at 37 °C. The medium was then removed, and the cells were treated with 300 nM syn-RA16 or scrambled RNA (SCAP) in fresh medium for series incubation time (0, 15, 30, 60, 120, 240, 480, and 960 min). After incubation, cells were rinsed three times with DPBS, and then collected for RNA extraction using TRIzol reagent (Thermo Fisher Scientific, Rockford, IL) according to the manufacturer's protocol. The extracted total RNAs were first treated with DNase I to eliminate DNA contamination and quantified using One Drop Spectrophotometry (Hong Kong, China). Next, 500 ng of DNase I-treated RNA was reverse-transcribed into DNA using M-MLV transcriptase (TaKaRa, Dalian, China). Real time PCR was performed with aptamer primers and Power SYBR Green Master Mix (Life Technologies) according to the manufacturer's protocol, and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH, primers sets from Sangon Technologies, Shanghai, China) was amplified for normalization. Quantitative PCR data were analyzed using the StepOnePlus ™ Real-Time PCR system (Applied Biosystems). The relative RNA levels with different incubation time were calculated by the 2 − ΔΔCT method using GAPDH as a control.
Cell viability assay. NCI-H460 or other cells were seeded in 96-well plates at 5 × 10 3 cells per well overnight.
The medium was then removed, and the cells were treated with RNA molecules in fresh medium at different concentrations. After 48 h of incubation, cell viability was determined using Cell Counting Kit-8 (CCK-8; Dojindo, Tokyo, Japan). The absorbance was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, Rockford, IL).
In vivo imaging analysis. After the tumor grew to 200~300 mm 3 , tumor-bearing mice were administered with Cy5.5-labeled RNA molecules by tail vein injection. Cy5.5-labeled RNA molecules were tracked using an in vivo imaging system (Kodak FX Pro; Carestream Health, Rochester, NY) at 0.5, 2, and 3.5 h post-injection 18 . Tumors were then exacted for imaging after 4 h of circulation.
In vivo trap assay with qRT-PCR. Three mice were administered with 1 nmol syn-RA16 or trans-RA16 via intravenous injection. After 3.5 h of circulation, tumor, heart, liver, lung, and kidney tissues were collected for RNA extraction using TRIzol reagent (Thermo Fisher Scientific, Rockford, IL) according to the manufacturer's protocol. The resulting total RNA was first treated with DNase I to eliminate DNA contamination and quantified using One Drop (Hong Kong, China). Next, 500 ng of DNase I-treated RNA was reverse-transcribed into DNA using M-MLV transcriptase (TaKaRa, Dalian, China). qPCR was performed with RA16 aptamer primers and Power SYBR Green Master Mix (Life Technologies) according to the manufacturer's protocol, and mouse 18S RNA (primers sets from Sangon Technologies, Shanghai, China) was amplified for normalization 18 . qPCR data were analyzed using the StepOnePlus ™ Real-Time PCR system (Applied Biosystems). The relative RNA levels in various tissues were calculated by the 2 −ΔΔCT method using mouse 18S RNA as the control 46,47 . Truncation of RA16 aptamers. RA16 aptamers were truncated into three parts. S1: 5′-GGGUGCCAAGCCGUCGGGUUAUGUUGAUCUCCUCAAGGAC-3′. S 2: 5 ′-GG GU GC CA AG CC G U CG GG UU AU GU UG AUCU CC UC AA GG AC GA GU GCAUUGCAUCACGUCAGUAG-3′ S3: 5′-GGGAGAGAA CAAUGACCUGCGGUGCCAAGCCGUCGGGUUAUGUUGAUCUCCUCAAGGACGAGUGCAUUG-3′. S1 was transcribed from the DNA sequence amplified by PCR using 5′-CACTAATACGACTCACTATAGG GTGCCAAGCCGTCGGGTTATGTTGATCTCCTCAAGGACGAGTGCATTGCATCACGTCAGTAG-3′ as template and the underlined sequences as primers.
S2 was transcribed from the DNA sequence amplified by PCR using 5′-CACTAATACGACTCACT ATAGGGTGCCAAGCC GTCGGGTTATGTTGATCTCCTCAAGGACGAGTGCATTGCATCACGTCAGTAG-3′ as template and the underlined sequences as primers.
S3 was transcribed from the DNA sequence amplified by PCR using 5′-CACTAATACGACTCACTATAGGGAGAG AACAATGACCTGCGGTGCCAAGCCGTCGGGTTATGTTGATCTCCACAAGGACGAGTGCATTG CATCACGTCAGTAG-3′ as template and the underlined sequences as primers.
The in vitro transcription process was similar to that described for the full-length aptamer with the incorporation of 16-biotin-UTP. The secondary structures of the truncated aptamers, which include the possible binding region, were predicted by Mfold software (http://unafold.rna.albany.edu/?q=mfold).

Statistical analysis.
Results are presented as the mean ± standard deviation of at least three independent experiments with duplicate samples. Statistical differences were evaluated using one-way analysis of variance unless otherwise indicated. P < 0.05 was considered as statistically significant. Graphs were generated by GraphPad Prism (version 6; GraphPad, La Jolla, CA, USA) and Microsoft Excel (version 2010).