A Novel ssDNA Aptamer Targeting Carcinoembryonic Antigen: Selection and Characterization

Abstract

One of the major causes of a drastically shorter life expectancy and one of the most prevalent diseases in the world today is cancer. Given the data on the rise in cancer cases throughout the world, it is obvious that, despite the diagnostic techniques currently being used, there is a pressing need to develop precise and sensitive techniques for early diagnosis of the disease. A high degree of affinity and specificity towards particular targets is maintained by the short nucleic acid molecules known as aptamers. Aptamers outperform antibodies due to their unique benefits, such as their simplicity in synthesis and modification, lack of toxicity, and long-term stability. Utilizing an accurate recognition element and a robust signal transduction mechanism, molecular diagnostics can be extremely sensitive and specific. In this study, development of new single-stranded DNA aptamers against CEA for use in cancer diagnostics was accomplished using SELEX and NGS methods. As a result of 12 iterative SELEX rounds, nine aptamer candidates against CEA were developed. NGS comparative analysis revealed that round twelve had an enriched number of aptamers that were specifically bound, as opposed to round eight. Among the selected nine sequences characterized by bioinformatics analysis and ELONA, an aptamer sequence with the highest specificity and affinity for the target protein was identified and further examined. Aptamer sequence (6) was screened in a concentration-dependent assay, specificity analysis was performed, and its potential secondary and tertiary structures were predicted, which enabled us to test one of the possible putative interactions with CEA. Finally, aptamer sequence (6) labelled with a Cy5 fluorescent tag was used in confocal microscopy to observe its binding towards the CEA expressed in HT-29 human colon adenocarcinoma cell line.

Rapid detection of carcinoembryonic antigen by means of an electrochemical aptasensor

“…The high sensitivity and specificity of the EIS aptasensor developed in this study were primarily attained through the utilization of the CEA aptamer sequence (6), selected and characterized in our previous study. 26 Adding linkers and five thymine residues to the aptamer created space between it and the surface, facilitating its folding and enhancing its ability to bind to the protein. 37 , 38 Ethylene oxide (CH 2 CH 2 O), a hydrophilic component of the linker, was crucial in extending the aptamer away from the surface monolayer, thereby preventing steric interference with protein binding.…”

“…The bare IDE surface was first modified using a thiolated CEA aptamer (6) sequence, which our research team previously selected and characterized. 26 In the current study, the ssDNA aptamer sequence (6) with a total length of 39 nucleotides underwent modification with the addition of a linker in the form of HS(CH 6 ) 6 -OP(O) 2 O-(CH 2 CH 2 O) 6 -5′-TTTTT- aptamer (6) -3′. Following this step, the IDE surface was backfilled with 6-mercapto-1-hexanol (MCH) to act as a co-immobilizing agent, thereby reducing non-specific binding.…”

Semi-automated and efficient parallel SELEX of aptamers for multiple targets

“…To better understand the enrichment of sequences in the affinity library, we performed high-throughput sequencing of the enriched library, the extracted data were compared for the frequency of each round, and then the top 300 sequences were arranged by family using ClustalX software, 22 and ssDNAs of different families were synthesized to obtain high-affinity aptamers. It can be seen from the figure that taking ROR1 02 and ROR1 15 targeting aptamers as an example, in the four rounds of SELEX, it is gradually enriched, and a progressively increasing number of candidate sequences in each round were selected when the sequences were synthesized (Fig.…”

Circulating Tumor Cells Adhesion: Application in Biosensors

“…These approaches enable specific interactions, are easily developed and modified, detect a variety of targets, and exhibit stability, biocompatibility, and reusability [ 129 , 133 , 134 ]. Aptamers have been extensively studied for CTC detection and the identification of new cancer biomarkers [ 63 , 135 , 136 , 137 , 138 ]. Moreover, these recognition ligands can amplify the sensor response signal, further enhancing their utility [ 52 , 60 , 61 , 117 , 129 ].…”