Fig.4.3: Cyclic voltammograms corresponding to the analysis of different concentrations of ER alpha protein (from to bottom) (a) 1mM, (b) 10 mM, (c) 25 mM. Fig.4.4: SWV plot showed the decrease in faradaic current when analyte ER alpha was added.
Introduction
Breast cancer treatment has undergone several changes in recent decades due to the discovery of specific prognostic and predictive biomarkers that allow the application of more individualized therapies to different molecular subgroups. (Williams et al. 2006) Overexpression of proteins can occur in patients with breast cancer. biomarkers are associated with poor prognosis, aggressiveness of the disease, as well as resistance to chemotherapy and hormonal therapy. Thus, strategies to target these protein biomarkers (HER2, ER and PR) are important in the treatment of advanced breast cancer (Esteva et al. 2002).
Biomarkers and Disease management
Although biomarkers allow easy epidemiological research, they are also useful in investigating the natural history and prognosis of a disease. For example, a biomarker will allow the stratification of a population based on a predisposed specific 'genotype' associated with a specific disease, rather than relying on a report of the 'family history' of the disease.
Biomarkers of Breast cancer
HER2, a transmembrane protein of the human epidermal growth factor receptor (hEGFR) family is a crucial predictive biomarker for various carcinomas (Tetu et al. 1996). Ismail et al. 2003) Regardless of any hypothesis, the need of the hour is to conduct more basic research to address the fundamental role of progesterone in mammary development and tumorigenesis.
Triple Negative Breast Cancer
The assessment of biomarkers by traditional monoclonal antibodies has faced a major challenge in recent years. These short, chemically synthesized, single-stranded (ss) RNA or DNA oligonucleotides fold into specific three-dimensional (3D) structures with dissociation constants typically in the pico- to nanomolar range.
Aptamers-A Novel class of oligonucleotides for disease management 18
As a result, the aptamer will retain its structure and function in the final assay. In the decade or so since the SELEX procedure was first introduced, a large literature has emerged (Brody et al. 1999;
Various SELEX Process
All previously described SELEX processes are based on prior knowledge of the target for aptamer selection. In this technology, aptamers are selected against an enantiomeric form of the target via the standard SELEX protocol.
Aptamers as Therapeutics
Keefe etc. 2010 ) AS1411 (formerly ARGO100; Antisoma) is a quadruple guanine aptamer obtained from a guanine-rich oligonucleotide library in the anti-proliferation screen, which is not a typical SELEX process. A subsequent phase II trial in renal cell carcinoma was initiated in 2008 (clinical trial ID NCT00740441). (Rosenberg et al. 2014) NOXA12 (Olaptesed pegol;.
Nucleic acid Based Biosensors
Scientists have recently developed supramolecular ionic liquids grafted onto nitrogen-doped graphene airgels (SIL-g-(N)GAs), which are used as a novel electrochemical DNA biosensor for the direct detection of BRCA1 gene-associated with breast cancer. In another study, another group developed an electrochemical DNA biosensor for the rapid detection of the sequence (5' AAT GGA . TTT ATC TGC TCT TCG 3') specific for breast cancer gene 1 (BRCA1).
Aptamer based Biosensors
This also testifies to a high selectivity and sensitivity for the detection of the tumor markers, which offers enormous potential for clinical applications in the future. (Zhao et al. 2012). Thus, it would provide a general model for the detection of tumor markers based on an impedimetric aptasensor.
Conclusion
Brandenberger AW, Tee MK, Lee JY, et al (1997) Tissue distribution of estrogen receptor alpha (ER-alpha) and beta (ER-beta) mRNA in the human fetus at mid-gestation. Cheang MCU, Chia SK, Voduc D, et al (2009) Ki67 index, HER2 status and prognosis of patients with luminal B breast cancer. Cheng C, Chen YH, Lennox K a, et al (2013) In vivo SELEX for Identification of Brain Penetrating Aptamers.
Esteva FJ, Sahin AA, Cristofanilli M, et al (2002) Molecular Prognostic Factors for Breast Cancer Metastasis and Survival. Gillett C, Fantl V, Smith R, et al (1994) Amplification and overexpression of cyclin D1 in breast cancer detected by immunohistochemical staining. Huang B, Omoto Y, Iwase H, et al (2014) Differential expression of estrogen receptor α, β1 and β2 in lobular and ductal breast cancer.
Liu G, Mao X, Phillips JA, et al (2009) Aptamer - nanoparticle strip biosensor for sensitive detection of cancer cells. Sun MQ, Cao FQ, Hu XL, et al (2014) [DNA aptamer selection in vitro for the determination of ketamine by FluMag-SELEX].
Selection and Characterization of Aptamers Specific To Estrogen
Outline of the Research Study
Experimental Section
An oligonucleotide library of 76 nt length, consisting of a central random region 40 nt long and flanking region of 18 nt at both 5' and 3' ends (5' ATACCAGTCTATTCAATT–N40- AGATAGTATGTGCAATCA 3'), forward primer ( 5'-ACCAGTTATTCAATTCAATCATTATCAAT -3'.conjugated with 6-FAM at 5'-end) and reverse primer (5'-TGATTGCACATACTATCT-3'.conjugated with biotin at 5'-end) were purchased from IDT Technologies (USA). The aptamer library was synthesized at 1 µM synthesis scale and PAGE purified while labeled and unlabeled primers were synthesized at 100 nM synthesis scale. To find the most likely ER α-aptamer, we performed preliminary analyzes such as free energy comparisons and structural stability of the selected candidates. Typically, 30 serial injections (1.5 µL per one injection) of 20 µM aptamer candidate with a spacing of 130 sec. made with continuous stirring of the solution (at 210 rpm) in the sample cell.
To determine the affinity of the aptamer-ERα complex, an assay for ligand-receptor interactions was performed. (Xu et al. Then, the purified recombinant ER α protein was coated in 96-well microtiter plates (2 μg/well) and incubated with increasing concentrations (0-1000nM) of biotinylated ER_Apt1. The cellular stability of the selected aptamer was determined by MTT dye (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide) conversion assay.
To evaluate the binding properties of the candidate aptamer to the ERα structure, we incubated ERα-positive MCF7 cells with FAM-tagged ER_Apt1 and tagged aptamer in 200 μl of binding buffer at 37 °C for 30 min. After thorough washing in 0.1 M PBS solution, antigenic epitopes of the tissue sections were visualized using a streptavidin-HRP conjugate (1:500) (S911, Invitrogen) and DAB plus substrate system (Invitrogen).
Results
But when ER_Apt1 was treated with ER beta, the band intensity did not change much. 6-FAM-labeled ER_Apt1 was co-incubated with 24-h cultured ERα-positive and negative cell lines. However, when incubated with the ERα-negative cell line MDA MB 231, FAM-tagged ER_apt1 resulted in a fluorescent signal comparable to treatment with random oligonucleotides.
Dot plot of SSC-H vs. FL1-H shows that treatment with different concentrations of FITC-ER_Apt1 does not change the granularity of ERα positive cell line MCF7 (i-ii) and ERα negative cell line MDA MB231 (iii-iv). The detection area of the aptamer-based protein detection assay was quantified by plotting a standard curve for ER_Apt1 in the working range of 0–1000 nM. Binding of enriched ER_Apt1 aptamer (0-1000 nM) to immobilized ERα (2 μg) was measured by colorimetric binding saturation assays.
L929, MDA MB231, MCF7 cells treated with biotinylated ER_Apt1 (D, E, F), biotinylated splice sequence as negative control (G, H, I) and with ER-specific mAb as positive control respectively (J, K, L). In a cell-based immunofluorescence assay, the FAM-tagged candidate ER_apt1 bound specifically to the nuclei of MCF7 cells.
Discussion
The dissociation constant (Kd) of the ER_Apt1-protein complex was found to be in the nM range, which was confirmed by ITC assays. The specific candidate ER_Apt1 was labeled with 6-FAM at the 5' end for its fluorescence study. These results indicated that ER_Apt1 binds specifically to ERα-containing cell lines but not to others.
Despite the structural complexity of cells or even tissues, ER_Apt1 was found to bind ERα precisely and sensitively. ER_Apt1 was labeled with biotin at the 5' end to determine the binding ability at the cell and tissue level. Furthermore, this study also proves that labeling ER_Apt1 with biotin (chemical) or FAM (fluorescence) does not alter its specificity towards its target ERα protein.
In paraffin-embedded tissue sections, biotinylated ER_Apt1 showed specific and ER alpha-selective localization in the nuclei of malignant ductal cancer cells. Overall, ER_Apt1 showed more potency than an ERα-specific monoclonal antibody as a diagnostic agent against ER alpha malignancies.
Conclusion
Lee JH, Shin SK, Jiang Y, et al (2015) Facilitated Tau Degradation by USP14 Aptamers via Enhanced Proteasome Activity. Liu Z, Duan J-H, Song Y-M, et al (2012) Novel HER2 Aptamer Selectively Delivers Cytotoxic Drug to HER2-Positive Breast Cancer Cells in Vitro. Petersen OW, Høyer PE, van Deurs B (1987) Frequency and distribution of estrogen receptor-positive cells in normal, non-lactating human breast tissue.
Shui B, Ozer A, Zipfel W, et al (2012) RNA aptamers that functionally interact with green fluorescent protein and its derivatives. Tonetti DA, Jordan VC (1999) The estrogen receptor: a logical target for breast cancer prevention with antiestrogens. Xu D, Chatakonda V, Kourtidis A, et al (2014) In search of new drug target sites on estrogen receptors using RNA aptamers.
Selection and Characterization of Aptamers Specific To The Extra
Outline of the Research Study
Experimental Section
Results
Discussion
Conclusion
Aptamer Based Electrochemical Sensor System For Detection of
- Outline of the Research Study
- Experimental Section
- Results and Discussion
- Conclusion
- References
This chapter describes a relatively general approach to aptamer-based electrochemical sensors, which are based on the conformational change of aptamers induced by target binding. Aptamers were attached to gold electrode surfaces by thiol chemistry, and then analytes were detected by an electrochemical apparatus.
Summary and Future Prospects
Future Prospects of this Study
We developed an aptamer-based electrochemical detection system for Haar 2- and ER-alpha-positive breast carcinomas. We have successfully demonstrated the specificity and sensitivity of specific aptamers at the in vitro level and validated in breast cancer tissue samples. With more focus on in vivo studies for potential clinical applications, aptamers can be developed in combination with DNA nanostructures, nanomaterials, or microfluidic devices as diagnostic probes or therapeutic agents for cancers and various other diseases.
To realize the full potential, aptamers can be exploited to develop diagnostic kits that will be more portable, cost-effective and robust using simple devices for real-time and on-site detection and monitoring, instead of the cumbersome and time-consuming diagnostic tests currently available in clinical laboratories. In terms of a therapeutic approach, there is still untapped potential in the combination of target recognition and strong binding properties of aptamers with exquisitely designed nanomaterials that can be used as effective platforms for drug delivery. Many nanomaterials, such as liposomes, polymer vesicles, and silica nanoparticles, combined with DNA aptamers have shown feasibility for use in in vivo targeted drug delivery.
Although they have enormous potential for these theranostics, tailored modifications and validation experiments are needed before aptamer-based drug delivery can reach clinical trials and ultimately the daily treatment of cancer patients. In vitro selection of DNA aptamers against extracellular domain of human epidermal growth receptor 2 in HER2-positive carcinomas.
