The application of Aptamer in biomarker discovery

Abstract

Biomarkers are detectable molecules that can reflect specific physiological states of cells, organs, and organisms and therefore be regarded as indicators for specific diseases. And the discovery of biomarkers plays an essential role in cancer management from the initial diagnosis to the final treatment regime. Practically, reliable clinical biomarkers are still limited, restricted by the suboptimal methods in biomarker discovery. Nucleic acid aptamers nowadays could be used as a powerful tool in the discovery of protein biomarkers. Nucleic acid aptamers are single-strand oligonucleotides that can specifically bind to various targets with high affinity. As artificial ssDNA or RNA, aptamers possess unique advantages compared to conventional antibodies. They can be flexible in design, low immunogenicity, relative chemical/thermos stability, as well as modifying convenience. Several SELEX (Systematic Evolution of Ligands by Exponential Enrichment) based methods have been generated recently to construct aptamers for discovering new biomarkers in different cell locations. Secretome SELEX-based aptamers selection can facilitate the identification of secreted protein biomarkers. The aptamers developed by cell-SELEX can be used to unveil those biomarkers presented on the cell surface. The aptamers from tissue-SELEX could target intracellular biomarkers. And as a multiplexed protein biomarker detection technology, aptamer-based SOMAScan can analyze thousands of proteins in a single run. In this review, we will introduce the principle and workflow of variations of SELEX-based methods, including secretome SELEX, ADAPT, Cell-SELEX and tissue SELEX. Another powerful proteome analyzing tool, SOMAScan, will also be covered. In the second half of this review, how these methods accelerate biomarker discovery in various diseases, including cardiovascular diseases, cancer and neurodegenerative diseases, will be discussed.

Keywords: Aptamer; Biomarker discovery; Cardiovascular diseases; Neurodegeneration-related diseases; SELEX; SOMAScan; cancer.

Fig. 1

An aptamer binds to the target. Single-stranded aptamer is folded to be functional. It binds to its target whether protein, RNA or cell, via conformational recognition

Fig. 2

The general procedure of SELEX. (1) The library is incubated with a negative target, and the unbound sequences are collected; (2) The unbound sequences are incubated with a positive target, and the bound sequences ate collected; (3) The bound sequences are amplified for the next round of SELEX.

Fig. 3

Scheme of secretome SELEX [34]. The initial library was first incubated with normal cell secretome. Aptamers that do not bind to the normal cell secretome were collected and proceeded to positive selection. After incubation with cancer cell secretome, bound aptamers were collected for further rounds of selection. After several rounds of selection, sequences of aptamers that bind to cancer cell secretome were analyzed by NGS.

Fig. 4

Workflow of Adaptive Dynamic Artificial Poly-ligand Targeting (ADAPT)[42]. The blood plasma samples are collected and incubated with the profiling library. Then the ssDNA-partitioning is performed for individual patient profile analysis

Fig. 5

Illustration of tissue-SELEX procedure [55]. (1) The library is incubated with the negative target and the unbound sequences are collected; (2) The unbound sequences are incubated with the positive target and the bound sequences ate collected; (3) The bound sequences are amplified by PCR amplification; (4) The dsDNA of PCR product is denatured and desalted for next round of SELEX.

Fig. 6

Schematic representation of SOMAScan. (1) The labeled SOMAmers are incubated with prepared samples to bind with proteins, and then the binding complexes are tagged with biotin (B) and fluorescent label (F); (2)The avidin beads (AB) are used to capture SOMAmers-protein complexes; (3)The biotin need to tag to the proteins; (4)The ultraviolet light (hv) is utilized to cleave the photocleavable group in order to release the complexes; (5) Polyanionic competitors (PC) are introduced to remove the nonspecific SOMAmers; (6)The primer bead (PB) facilitate the complexes capture; (7) Captured complexes are dissociated in specific high pH condition