Activatable Aptamer Probe Research Articles

A pH-Sensitive I-Motif Aptamer Probe for Dual Targeting and Imaging of Pancreatic Cancer Cells via Nucleolin Interactions

This study investigates the targeted imaging and tumor inhibition effects of a “signal-off” imaging-based activatable aptamer probe (I-AAP) for pancreatic cancer. AS1411, contained in the I-AAP probe, targets the nucleolin and i-motifs on the membrane surfaces of pancreatic cancer cells (PANC-1) and has a dual response to pH and an acidic environment. In vitro, a “signal-off” type I-AAP probe was constructed that possesses a fluorescence effect in a phosphate buffer solution at pH 6.0 and 6.5. The probe has no fluorescence effect at pH 7.0 or 7.5; thus, the “signal-off” type I-AAP probe has a targeted acid-sensitive response in pancreatic cancer and a tumor microenvironment imaging effect. After incubation for 30 min, AS1411 was taken up by PANC-1 cells and reached its peak value within 2 h. AS1411 inhibited pancreatic cancer cells in a time-dependent manner. This “signal-off” I-AAP probe is expected to be suitable for a low-background and high-specificity molecular pathologic tracer technique, making use of the presence of nucleolins on the surface of pancreatic cancer cells and the low pH response of the tumor microenvironment.

A multivalent activatable aptamer probe with ultralow background signal and high sensitivity for diagnosis of lung adenocarcinoma

Lung adenocarcinoma is the most common subtype of lung cancer. Developing a simple, rapid, and sensitive method for the detection of lung adenocarcinoma is helpful to reduce the clinical workload and facilitate clinical treatment. To detect lung adenocarcinoma cells, this study designed a multivalent activatable aptamer probe (multi-AP) using S1 aptamer that can specifically recognize A549 cells (a lung adenocarcinoma cell line). Firstly, the monovalent activatable aptamer probe (mono-AP) was designed by strand-displacement strategy and fluorescent resonance energy transfer (FRET). The multi-AP was constructed by mono-AP assembled on the nucleic acid scaffold obtained by hybridization chain reaction (HCR). The presence of the target cell activated the multi-AP, the complementary strand was released, and the fluorescence detection signal was reported based on the FRET. The multi-AP has good specificity and sensitivity for detecting A549 in different matrices and greatly reduces the background signal with the lowest detection limit of 6 cells/200 μL. Furthermore, the multi-AP could directly detect the lung adenocarcinoma cells in cytology specimens, indicating a good potential of the multi-AP in clinical cytology specimens for rapid diagnosis, such as rapid on-site cytological evaluation. Besides, the multi-AP was further modified with biotin (bio-multi-AP) and used to detect clinical cytology cell blocks, implying the value of bio-multi-AP in immunocytochemistry for accurate diagnosis of lung adenocarcinoma.

Multivalent Engineering of Exosomes with Activatable Aptamer Probes for Specific Regulation and Monitoring of Cell Targeting.

Reconstituting and probing exosome-cell interactions is critical for elucidating exosome-related cell biology and advancing their diagnostic and therapeutic potential. We report here an exosomal engineering strategy to achieve controlled regulation of exosome-cell interactions with activatable sensing capability. The approach relies on membrane-protein directed, programmable DNA self-assembly to construct a DNA polymeric scaffold with multivalent display of structure-switchable aptamer sensing probes on exosome surfaces. The engineered exosomes exhibit enhanced cancer cell targeting ability compared to exosomes modified with monovalent aptamers. Furthermore, the anchored aptamer probes could be activated by specific membrane protein targeting, followed by structural switching to report an output fluorescence signal, thus allowing dynamic monitoring of exosome-cell interactions both in vitro and in vivo. We envision this will provide a complementary tool for specific regulation and monitoring of exosome-cell docking interactions and will advance the development of exosome-based biomedical applications.

Precise monitoring of mesenchymal stem cell homing to injured kidney with an activatable aptamer probe generated by cell-SELEX

Deficiency of precise tracking tools to monitor the migration and survival status of mesenchymal stem cells (MSCs) in the animal body has bottlenecked the clinical translation of stem cell therapies. In this study, we have for the first time successfully screened an aptamer, termed seq3, with high specificity and affinity for mouse bone marrow MSCs (mBMSCs) by MSC-based systematic evolution of ligands by exponential enrichment (MSC-SELEX), and ingeniously designed seq3-based activatable aptamer probes (AAP) for live-cell tracking. As proof of concept, real-time monitoring of transplanted mBMSCs in a mouse model of chronic kidney disease (CKD) was performed with the AAPs, and compared with Fe3O4-based magnetic resonance imaging (MRI), a widely utilized cell imaging module. It was confirmed that seq3 specifically binds to mBMSCs in the animal body and the binding activates the fluorescence of otherwise quenched dyes in AAPs, substantially minimizing the background signals originating from non-target tissues and avoiding the presence of false positive and/or false negative results occurred in MR imaging. Furthermore, the seq3-AAP-based fluorescence imaging was advantageous over Fe3O4-based MR imaging in terms of indicating survival status of the migrating and colonized mBMSCs, critical information required to evaluate the effectiveness and efficiency of cell therapy strategies. AAP fluorescence imaging results showed that the transplanted mBMSCs were well distributed in the bodies of normal mice and mainly aggregated in the livers, while they tended to migrate to injured kidneys of CKD mice and facilitated the repair of kidneys as a result. Besides cell tracking, the high specificity and affinity of seq3 to mBMSCs can be widely explored to understand the fate and biological roles of stem cells in regenerating tissues in the future.

A Simple, pH-Activatable Fluorescent Aptamer Probe with Ultralow Background for Bispecific Tumor Imaging.

Activatable aptamer probes (AAPs) are promising in molecular imaging of tumors, but the reported shape-switching-dependent AAPs are still challenged by unsatisfied noise suppression, poor stability, and sophisticated sequence design. To address the problem, we constructed a pH-activatable aptamer probe (pH-AAP) by utilizing an acid-labile acetal linker as the responsive element to be fused with a tumor-targeted aptamer. Specifically, a Cy5-labeled aptamer was connected with the quencher BHQ2 through the acetal group, thus generating pH-AAP with quenched fluorescence. Due to the stable proximity of Cy5 to BHQ2, pH-AAP was found to have ultralow background with a quenching efficiency as high as 98%. In comparison with shape-switching-dependent AAPs, the noise suppression of pH-AAP was well maintained for a much longer time in both serum and mouse body, thus showing a robust fluorescence stability. By a combination of the fluorescence recovery induced by acid hydrolysis of acetal linkers and the tumor-targeted recognition of aptamers, pH-AAP could either specifically anchor the extracellular pH-activated signals on the target cell surface in an acidic tumor microenvironment or be activated by acidic lysosomes after it was internalized into target cells. As proof of concept, in vitro evaluation and in vivo imaging of A549 lung cancer cells were performed by using S6 aptamer as a demonstration. It was indicated that pH-AAP realized washing-free, bispecific, and contrast-enhanced tumor imaging. The strategy is simple and free of sequence modification, which promises to provide a universal platform for sensitive and precise tumor diagnosis.

Molecular-Recognition-Based DNA Nanodevices for Enhancing the Direct Visualization and Quantification of Single Vesicles of Tumor Exosomes in Plasma Microsamples.

Tumor exosomes (Exo) are presumed to expedite both the growth and metastasis of tumors by actively participating in nearly all aspects of cancer development. Tumor-derived Exos are thus proposed as a resource for diagnostic biomarkers in bodily fluids. However, most Exo assays require large samples and are time-consuming, complicated, and costly, and thus unsuited for practical applications. Herein, we show an ultrasensitive assay that can directly visualize and quantify tumor Exos in plasma microsamples (1 μL) at the single-vesicle level. The assay uses the specific binding of activatable aptamer probes (AAP) to target Exos captured by Exo-specific antibodies on the surface of a flow cell to produce activated fluorescence. Furthermore, the bound AAP triggers in situ assembly of a DNA nanodevice with enhanced fluorescence that improves the Exo-detection sensitivity. By identifying tyrosine-protein-kinase-like 7 (PTK7), a total-internal-reflection-fluorescence (TIRF) assay for PTK7-Exo distinguishes target tumors from control subjects. This assay is also informative in monitoring tumor progression and early responses to therapy. The developed assay can be readily adapted for diagnosis and monitoring of other disease-associated Exo biomarkers.

DNA nanotriangle-scaffolded activatable aptamer probe with ultralow background and robust stability for cancer theranostics

Activatable aptamers have emerged as promising molecular tools for cancer theranostics, but reported monovalent activatable aptamer probes remain problematic due to their unsatisfactory affinity and poor stability. To address this problem, we designed a novel theranostic strategy of DNA nanotriangle-scaffolded multivalent split activatable aptamer probe (NTri-SAAP), which combines advantages of programmable self-assembly, multivalent effect and target-activatable architecture.Methods: NTri-SAAP was assembled by conjugating multiple split activatable aptamer probes (SAAPs) on a planar DNA nanotriangle scaffold (NTri). Leukemia CCRF-CEM cell line was used as the model to investigate its detection, imaging and therapeutic effect both in vitro and in vivo. Binding affinity and stability were evaluated using flow cytometry and nuclease resistance assays.Results: In the free state, NTri-SAAP was stable with quenched signals and loaded doxorubicin, while upon binding to target cells, it underwent a conformation change with fluorescence activation and drug release after internalization. Compared to monovalent SAAP, NTri-SAAP displayed greatly-improved target binding affinity, ultralow nonspecific background and robust stability in harsh conditions, thus affording contrast-enhanced tumor imaging within an extended time window of 8 h. Additionally, NTri-SAAP increased doxorubicin loading capacity by ~5 times, which further realized a high anti-tumor efficacy in vivo with 81.95% inhibition but no obvious body weight loss.Conclusion: These results strongly suggest that the biocompatible NTri-SAAP strategy would provide a promising platform for precise and high-quality theranostics.

A pH-responsive activatable aptamer probe for targeted cancer imaging based on i-motif-driven conformation alteration

We present here a pH-responsive activatable aptamer probe for targeted cancer imaging based on i-motif-driven conformation alteration. This pH-responsive activatable aptamer probe is composed of two single-stranded DNA. One was used for target recognition, containing a central, target specific aptamer sequence at the 3'-end and an extension sequence at the 5'-end with 5-carboxytetramethylrhodamine (TAMRA) label (denoted as strand A). The other (strand I), being competent to work on the formation of i-motif structure, contained four stretches of the cytosine (C) rich domain and was labeled with a Black Hole Quencher 2 (BHQ2) at the 3'-end. At neutral or slightly alkaline pH, strand I was hybridized to the extension sequence of strand A to form a double-stranded DNA probe, termed i-motif-based activatable aptamer probe (I-AAP). Because of proximityinduced energy transfer, the I-AAP was in a “signal off” state. The slightly acidic pH enforced the strand I to form an intramolecular i-motif and then initiated the dehybridization of I-AAP, leading to fluorescence readout in the target recognition. As a demonstration, AS1411 aptamer was used for MCF-7 cells imaging. It was displayed that the I-AAP could be carried out for target cancer cells imaging after being activated in slightly acidic environment. The applicability of I-AAP for tumor tissues imaging has been also investigated by using the isolated MCF-7 tumor tissues. These results implied the I-AAP strategy is promising as a novel approach for cancer imaging.

Designing activatable aptamer probes for simultaneous detection of multiple tumor-related proteins in living cancer cells

We report a novel strategy for high specific and simultaneous detection of multiple tumor-related proteins in cancer cells based on the activated fluorescence signal, which is triggered by specific-binding-induced conformation alteration of the designed activatable aptamer probe. The activatable aptamer probe consists two fragments: a target-protein-recognized aptamer sequence for specifically recognizing the protein, and an extending spacer making the aptamer in hairpin structure to enable the close proximity of quencher to fluorophore, which is labeled at 5′- and 3′-terminus, respectively, of the probes. Before interaction with cell, the fluorescence of the probe is quenched due to fluorescent resonance energy transfer (FRET) between the fluorophore and quencher. After interaction events, the fluorescence signal is activated through specific binding of the probe with target protein in cell, causing the conformation alteration and forcing the separation of fluorophore from the quencher. We achieve simultaneous detection of multiple tumor-related proteins in cells by designing the different activatable aptamer probes with various fluorophore/quencher combinations. Moreover, it can also achieve a high detection sensitivity (for example, detecting MCF-7 cells at a low abundance of ~(10±5)cellsmL−1) and specific discrimination of the subtype of cancers. The advantage of this approach is that it has high detection sensitivity because of the significant suppression of background with use of the designed activatable aptamer probe. In addition, it has ability of avoiding false signals arising due to the nonspecific adsorption of interferents because it operates via monitoring the activated fluorescence signals of the designed activatable aptamer probe.

A versatile activatable fluorescence probing platform for cancer cells in vitro and in vivo based on self-assembled aptamer/carbon nanotube ensembles.

Activatable aptamer probes (AAPs) have emerged as a promising strategy in cancer diagnostics, but existing AAPs remain problematic due to complex design and synthesis, instability in biofluids, or lack of versatility for both in vitro and in vivo applications. Herein, we proposed a novel AAP strategy for cancer cell probing based on fluorophore-labeled aptamer/single-walled carbon nanotube (F-apt/SWNT) ensembles. Through π-stacking interactions and proximity-induced energy transfer, F-apt/SWNT with quenched fluorescence spontaneously formed in its free state and realized signal activation upon targeting surface receptors of living cells. As a demonstration, Sgc8c aptamer was used for in vitro analysis and in vivo imaging of CCRF-CEM cancer cells. It was found that self-assembled Cy5-Sgc8c/SWNT held robust stability for biological applications, including good dispersity in different media and ultralow fluorescence background persistent for 2 h in serum. Flow cytometry assays revealed that Cy5-Sgc8c/SWNT was specifically activated by target cells with dramatic fluorescence elevation and showed improved sensitivity with as low as 12 CCRF-CEM cells detected in mixed samples containing ~100,000 nontarget cells. In vivo studies confirmed that specifically activated fluorescence was imaged in CCRF-CEM tumors, and compared to "always on" probes, Cy5-Sgc8c/SWNT greatly reduced background signals, thus resulting in contrast-enhanced imaging. The general applicability of the strategy was also testified by detecting Ramos cells with aptamer TD05. It was implied that F-apt/SWNT ensembles hold great potential as a simple, stable, sensitive, specific, and versatile activatable platform for both in vitro cancer cell detection and in vivo cancer imaging.

Au@Ag/Au nanoparticles assembled with activatable aptamer probes as smart "nano-doctors" for image-guided cancer thermotherapy.

Although nanomaterial-based theranostics have increased positive expectations from cancer treatment, it remains challenging to develop in vivo "nano-doctors" that provide high-contrast image-guided site-specific therapy. Here we designed an activatable theranostic nanoprobe (ATNP) via self-assembly of activatable aptamer probes (AAPs) on Au@Ag/Au nanoparticles (NPs). As both quenchers and heaters, novel Au@Ag/Au NPs were prepared, showing excellent fluorescence quenching and more effective near-infrared photothermal therapy than Au nanorods. The AAP comprised a thiolated aptamer and a fluorophore-labeled complementary DNA; thus, the ATNP with quenched fluorescence in the free state could realize signal activation through target binding-induced conformational change of the AAP, and then achieve on-demand treatment under image-guided irradiation. By using S6 aptamer as the model, in vitro and in vivo studies of A549 lung cancer verified that the ATNP greatly improved imaging contrast and specific destruction, suggesting a robust and versatile theranostic strategy for personalized medicine in future.

A label-free activatable aptamer probe for colorimetric detection of cancer cells based on binding-triggered in situ catalysis of split DNAzyme.

A novel label-free tailed hairpin-shaped activatable aptamer probe (THAAP) was developed by rationally integrating an aptamer and a split G-quadruplex into one sequence. Based on target recognition-triggered in situ catalysis of split DNAzyme, the THAAP strategy achieved a simple, fast, washing-free, specific and quantitative colorimetric assay of human leukemic CCRF-CEM cells.

Recent advances in fluorescent nucleic acid probes for living cell studies

Living cell studies can offer tremendous opportunities for biological and disease studies. Due to their high sensitivity and selectivity, minimum interference with living biological systems, ease of design and synthesis, fluorescent nucleic acid probes (FNAPs) have been widely used in living cell studies, such as for intracellular detection, cell detection, and cell-to-cell communication. Here, we review the general requirements and the recent developments in FNAPs for living cell studies. We broadly classify these designs as hybridization probes and aptamer probes. For hybridization probes, we describe recently developed designs, such as nanomaterial-based and amplification-based hybridization probes. For aptamer probes, we discuss four general paradigms that have appeared most frequently in the literature: nanomaterial-based, nanomachine-based, cell surface-anchored and activatable aptamer probe designs in vivo. FNAPs promise to open up new and exciting opportunities in biological marks detection for a wide range of biological and medical applications.

Activatable aptamer probe for contrast-enhanced in vivo cancer imaging based on cell membrane protein-triggered conformation alteration

Aptamers have emerged as promising molecular probes for in vivo cancer imaging, but the reported "always-on" aptamer probes remain problematic because of high background and limited contrast. To address this problem, we designed an activatable aptamer probe (AAP) targeting membrane proteins of living cancer cells and achieved contrast-enhanced cancer visualization inside mice. The AAP displayed a quenched fluorescence in its free state and underwent a conformational alteration upon binding to target cancer cells with an activated fluorescence. As proof of concept, in vitro analysis and in vivo imaging of CCRF-CEM cancer cells were performed by using the specific aptamer, sgc8, as a demonstration. It was confirmed that the AAP could be specifically activated by target cancer cells with a dramatic fluorescence enhancement and exhibit improved sensitivity for CCRF-CEM cell analysis with the cell number of 118 detected in 200 μl binding buffer. In vivo studies demonstrated that activated fluorescence signals were obviously achieved in the CCRF-CEM tumor sites in mice. Compared to always-on aptamer probes, the AAP could substantially minimize the background signal originating from nontarget tissues, thus resulting in significantly enhanced image contrast and shortened diagnosis time to 15 min. Furthermore, because of the specific affinity of sgc8 to target cancer cells, the AAP also showed desirable specificity in differentiating CCRF-CEM tumors from Ramos tumors and nontumor areas. The design concept can be widely adapted to other cancer cell-specific aptamer probes for in vivo molecular imaging of cancer.