Selection of aptamers against triple negative breast cancer cells using high throughput sequencing

Ferreira, Débora; Barbosa, José; Sousa, D. Z.; Melo, Luís D. R.; Avci‐Adali, Meltem; Wendel, Hans Peter; Rodrigues, L. R.

doi:10.1038/s41598-021-87998-y

Cited by 24 publications

(8 citation statements)

References 41 publications

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“…The use of NGS for this purpose is highly advantageous over conventional Sanger DNA sequencing, since the number of possible reads can be increased from a few to millions. Using NGS, there is a much greater possibility of selecting highly specific aptamers from among those generated by cell-SELEX [ 39 , 45 ]. Further, when high throughput sequencing is combined with bioinformatics analysis, other information, besides a large pool of potential aptamer candidates, can be derived, such as the number of total reads, frequencies of every unique sequence and the rate of molecular enrichment [ 46 ].…”

Section: Resultsmentioning

confidence: 99%

“…The mfold web server (version 3.0, accessed on 17 July 2022) was used to calculate the Gibbs free energy (∆G) and secondary structures of the sequences considering the primer and random regions, presenting an estimate of the optimal structure for each aptamer. The conditions used for the structure predictions were set according to the SB constitution (137 mM Na + and 1.4 mM Mg 2+ at pH 7.4) at 37 °C [ 39 ]. These sequences were aligned and the phylogenetic tree was constructed using the Tree Builder function in Geneious, that allowed the determination of the tree distances and sequence relatedness using a neighbor-joining model.…”

Section: Methodsmentioning

confidence: 99%

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Electrochemical Aptasensor for the Detection of the Key Virulence Factor YadA of Yersinia enterocolitica

et al. 2022

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New point-of-care (POC) diagnosis of bacterial infections are imperative to overcome the deficiencies of conventional methods, such as culture and molecular methods. In this study, we identified new aptamers that bind to the virulence factor Yersinia adhesin A (YadA) of Yersinia enterocolitica using cell-systematic evolution of ligands by exponential enrichment (cell-SELEX). Escherichia coli expressing YadA on the cell surface was used as a target cell. After eight cycles of selection, the final aptamer pool was sequenced by high throughput sequencing using the Illumina Novaseq platform. The sequencing data, analyzed using the Geneious software, was aligned, filtered and demultiplexed to obtain the key nucleotides possibly involved in the target binding. The most promising aptamer candidate, Apt1, bound specifically to YadA with a dissociation constant (Kd) of 11 nM. Apt1 was used to develop a simple electrochemical biosensor with a two-step, label-free design towards the detection of YadA. The sensor surface modifications and its ability to bind successfully and stably to YadA were confirmed by cyclic voltammetry, impedance spectroscopy and square wave voltammetry. The biosensor enabled the detection of YadA in a linear range between 7.0 × 104 and 7.0 × 107 CFU mL−1 and showed a square correlation coefficient >0.99. The standard deviation and the limit of detection was ~2.5% and 7.0 × 104 CFU mL−1, respectively. Overall, the results suggest that this novel biosensor incorporating Apt1 can potentially be used as a sensitive POC detection system to aid the diagnosis of Y. enterocolitica infections. Furthermore, this simple yet innovative approach could be replicated to select aptamers for other (bacterial) targets and to develop the corresponding biosensors for their detection.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Electrochemical Aptasensor for the Detection of the Key Virulence Factor YadA of Yersinia enterocolitica

et al. 2022

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“…These predictions can provide not only information about the conformation of the secondary structure of aptamers but also information on Gibbs free energy to judge the stability of the structure formed by aptamers. Typically, more structurally stable aptamers are selected for aptamer truncation and further applications [ 58 ]. Several researches have shown that the loop regions of aptamers are useful in most aptamer-target interactions.…”

Section: Post-selex Optimization Of Aptamersmentioning

confidence: 99%

Systematic bio-fabrication of aptamers and their applications in engineering biology

Cai

Chen

Zhang

et al. 2022

Syst Microbiol and Biomanuf

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Aptamers are single-stranded DNA or RNA molecules that have high affinity and selectivity to bind to specific targets. Compared to antibodies, aptamers are easy to in vitro synthesize with low cost, and exhibit excellent thermal stability and programmability. With these features, aptamers have been widely used in biology and medicine-related fields. In the meantime, a variety of systematic evolution of ligands by exponential enrichment (SELEX) technologies have been developed to screen aptamers for various targets. According to the characteristics of targets, customizing appropriate SELEX technology and post-SELEX optimization helps to obtain ideal aptamers with high affinity and specificity. In this review, we first summarize the latest research on the systematic bio-fabrication of aptamers, including various SELEX technologies, post-SELEX optimization, and aptamer modification technology. These procedures not only help to gain the aptamer sequences but also provide insights into the relationship between structure and function of the aptamers. The latter provides a new perspective for the systems bio-fabrication of aptamers. Furthermore, on this basis, we review the applications of aptamers, particularly in the fields of engineering biology, including industrial biotechnology, medical and health engineering, and environmental and food safety monitoring. And the encountered challenges and prospects are discussed, providing an outlook for the future development of aptamers.

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“…DNA aptamers are single-stranded DNA oligonucleotides that, through their unique three-dimensional (3D) structures, can be used for both selective binding to specific target molecules and can also elicit a functional effect upon binding and thus represent a promising molecular tool in this arena. − Highly selective DNA aptamers are emerging as the most promising candidates for targeting the cell surface and intracellular target proteins. − They have gained increasing attention for cancer diagnosis and treatment because of their low molecular weight, lack of or low immunogenicity, versatility in terms of manipulation for improved stability, and target efficacy . Aptamers are generally isolated by the SELEX (Systematic Evolution of Ligands by EXponential enrichment) process. , SELEX is typically carried out for a single target protein in solution and by repeated rounds − of selection and enrichment using PCR amplification. One possible drawback of SELEX is inefficient enrichment of sequences, i.e., less-structured nonbinding sequences, that can be more easily amplified by PCR polymerases .…”

Section: Introductionmentioning

confidence: 99%

Novel DNA Aptamer for CYP24A1 Inhibition with Enhanced Antiproliferative Activity in Cancer Cells

Biyani

Yasuda

Isogai

et al. 2022

ACS Appl. Mater. Interfaces

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Overexpression of the vitamin D3-inactivating enzyme CYP24A1 (cytochrome P450 family 24 subfamily and hereafter referred to as CYP24) can cause chronic kidney diseases, osteoporosis, and several types of cancers. Therefore, CYP24 inhibition has been considered a potential therapeutic approach. Vitamin D3 mimetics and small molecule inhibitors have been shown to be effective, but nonspecific binding, drug resistance, and potential toxicity limit their effectiveness. We have identified a novel 70-nt DNA aptamer-based inhibitor of CYP24 by utilizing the competition-based aptamer selection strategy, taking CYP24 as the positive target protein and CYP27B1 (the enzyme catalyzing active vitamin D3 production) as the countertarget protein. One of the identified aptamers, Apt-7, showed a 5.8-fold higher binding affinity with CYP24 than the similar competitor CYP27B1. Interestingly, Apt-7 selectively inhibited CYP24 (the relative CYP24 activity decreased by 39.1 ± 3% and showed almost no inhibition of CYP27B1). Furthermore, Apt-7 showed cellular internalization in CYP24-overexpressing A549 lung adenocarcinoma cells via endocytosis and induced endogenous CYP24 inhibition-based antiproliferative activity in cancer cells. We also employed high-speed atomic force microscopy experiments and molecular docking simulations to provide a single-molecule explanation of the aptamer-based CYP24 inhibition mechanism. The novel aptamer identified in this study presents an opportunity to generate a new probe for the recognition and inhibition of CYP24 for biomedical research and could assist in the diagnosis and treatment of cancer.

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Selection of aptamers against triple negative breast cancer cells using high throughput sequencing

Cited by 24 publications

References 41 publications

Electrochemical Aptasensor for the Detection of the Key Virulence Factor YadA of Yersinia enterocolitica

Electrochemical Aptasensor for the Detection of the Key Virulence Factor YadA of Yersinia enterocolitica

Systematic bio-fabrication of aptamers and their applications in engineering biology

Novel DNA Aptamer for CYP24A1 Inhibition with Enhanced Antiproliferative Activity in Cancer Cells

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