A novel analytical probe binding to a potential carcinogenic factor of N-glycolylneuraminic acid by SELEX
Introduction
N-glycolylneuraminic acid (Neu5Gc) is an acidic 9-carbon sugar found primarily in deuterostome-lineage animals (Kawano et al., 1994, Chen and Varki, 2010, Varki, 1992). It plays an important role in cell-cell recognition and adherence, inflammation, immune response, and transformation of tumor cells (Yamakawa et al., 2007, Hedlund et al., 2007). However, Neu5Gc is not present in the tissues of chickens or healthy humans. The human deficiency of Neu5Gc is explained by an inactivating mutation in the gene encoding CMP-N-acetylneuraminic acid (Neu5Ac); this mutation is a 92-bp deletion corresponding to the loss of a single human exon homologous to exon 6 of the mouse hydroxylase gene, which was discovered after sequencing the entire corresponding region of the human genome (Muchmore et al., 1998, Varki, 2001, Varki, 2009, Malykh et al., 1998, Chou et al., 1998). However, the use of various immunological and chemical techniques has suggested that expression of Neu5Gc can be detected in some visceral cancers, such as liver, gastric and colorectal cancers (Morito et al., 1982, Devine et al., 1991, Nasonkin and Koliatsos, 2006).
Glycoconjugate-bound Neu5Gc is a common component of animal-based dietary sources, particularly in most types of red meat. Neu5Gc might be released by the action of sialidases present in the gut and could be detected in the human body following consumption and digestion of organisms that normally express Neu5Gc (Diaz et al., 2009, Hirabayashi et al., 1987, Taylor et al., 2010). However, the mechanisms of sialic acid resorption, its distribution throughout the body, its uptake by cells, and expression of its potential precursor, N-glycolylmannosamine (ManNGc), are not clearly known. It is reported that cultured human cells are capable of taking up free sialic acid from the medium and incorporating it into glycoconjugates in vitro. Thus, the possibility that Neu5Gc-containing glycoconjugates are taken up directly from the diet and to what extent dietary Neu5Gc-containing gangliosides can become conjugated to the sialic acids of human tissues remains to be investigated.
Immunodiagnosis of tumors is mainly based on a specific reaction between tumor antigen and antibody. As Neu5Gc is endogenously expressed in mammals, mice do not react strongly to Neu5Gc antigen, which is necessary for the preparation of monoclonal antibodies. The human anti-Neu5Gc antibodies are complex, polyclonal, and variable amongst individuals, and the Neu5Gc antigen can be of exogenous or endogenous origin. Indeed, both polyclonal and monoclonal antibodies against Neu5Gc have been raised in chickens and used to detect this sialic acid in tissue sections and lipid extracts from human tumors and fetuses. Although Neu5Gc can induce chickens to generate IgG or IgY immunoglobulins, the polyclonal antibody has a poor specificity. Therefore, the preparation of a specific antigenic probe is a bottleneck for development of accurate immunodiagnostics.
As an alternative to antibodies, aptamers have shown promising applications in diagnostics and therapeutics. Aptamers are ssDNA, RNA, or modified nucleic acids. They are screened from a chemically-synthesized nucleic acid combinatorial library by an in vitro selection process called “systematic evolution of ligands by exponential enrichment” (SELEX) (Tuerk and Gold, 1990, Jayasena, 1999). Because of their unique three-dimensional structures, aptamers can bind selectively and specifically to peptides, proteins, and pathogenic targets by hydrogen bonding, Van der Waals forces, hydrophobic effects and other mechanisms (Tuerk et al., 1992, Sassanfar and Szostak, 1993, Wilson et al., 1998, Macaya et al., 1993, Drolet et al., 1999). In contrast to antibodies, aptamers are non-immunogenic, consistent, animal-friendly, and small (5—15 kDa), and aptamers can show favorable target-to-objection ratios in a short time at a relatively low cost, allowing better batch-to-batch reproducibility and easier incorporation of chemical modifications. Aptamers, which are synthetic molecules, have emerged as being potentially applicable to the diagnosis and treatment of cancer by inhibiting tumor angiogenesis, blocking signal transduction, inhibiting tumor cell proliferation, promoting tumor cell apoptosis, inhibiting tumor metastasis and as immunotherapy treatments (Bock et al., 1992).
In this study, we report the first development of DNA aptamers that are able to bind Neu5Gc with high affinity and selectivity. The results presented in this report provide the first step in the development of an affordable, sensitive and high-throughput assay for establishing relatively easy and quick laboratory detection methods for Neu5Gc.
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Reagents and materials
The synthetic random DNA library, primers and biotin-labeled primers were synthesized by Shanghai Sangon Biological Engineering Technology & Services Company (Shanghai, China). Bovine serum albumin (BSA) was purchased from Beijing Dingguo Biotechnology Development Centre (Beijing, China). N-hydroxysuccinimide, N,N-dicyclohexylcarbodiimide (DCC), and N,N-dimethylformamide (N,N-DMF) were purchased from Sigma (CA, USA). Streptavidin conjugated to horseradish peroxidase was purchased from Beijing
Conjugation of Neu5Gc and immobilization on microtiter plates
The active ester of Neu5Gc was prepared by two general approaches: activation of the acid by routine procedures followed by reaction with the hydroxyl compound HOR2, which may be present during the activation or added afterwards; and transesterification of the oxy moiety of OR2 from the carbonic acid ester to the Neu5Gc. A schematic of this process is shown in Fig. 1A.
The Neu5Gc-BSA conjugates were analyzed by agarose gel electrophoresis and UV spectrometry. As shown in Fig. 1B, the Neu5Gc-BSA
Conclusions
In this study, we report the development of aptamers specific for Neu5Gc for the first time. The 6 different aptamers selected for affinity for Neu5Gc were investigated by using ELISA and direct competitive ELISA. Of these 6 aptamers, N8 showed the best IC50 value (127 ng mL−1) and had a relatively high affinity constant (Ka=6.68×109 M−1). Due to its high sensitivity and specificity, the aptamers selected in this study can be used as a recognition element for the development of Neu5Gc detection
Acknowledgements
The authors are thankful for the financial support of the National Nature Science Foundation of China (NSFC, No. 31071539) and the Fundamental Research Funds for Jilin University (No.421060536206).
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- Both authors contributed equally to this work.
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