Novel system for detecting SARS coronavirus nucleocapsid protein using an ssDNA aptamer

Abstract

The outbreak of severe acute respiratory syndrome (SARS) in 2002 affected thousands of people and an efficient diagnostic system is needed for accurate detection of SARS coronavirus (SARS CoV) to prevent or limit future outbreaks. Of the several SARS CoV structural proteins, the nucleocapsid protein has been shown to be a good diagnostic marker. In this study, an ssDNA aptamer that specifically binds to SARS CoV nucleocapsid protein was isolated from a DNA library containing 45-nuceotide random sequences in the middle of an 88mer single-stranded DNA. After twelve cycles of systematic evolution of ligands by exponential enrichment (SELEX) procedure, 15 ssDNA aptamers were identified. Enzyme-linked immunosorbent assay (ELISA) analysis was then used to identify the aptamer with the highest binding affinity to the SARS CoV nucleocapsid protein. Using this approach, an ssDNA aptamer that binds to the nucleocapsid protein with a K(d) of 4.93±0.30nM was identified. Western blot analysis further demonstrated that this ssDNA aptamer could be used to efficiently detect the SARS CoV nucleocapsid protein when compared with a nucleocapsid antibody. Therefore, we believe that the selected ssDNA aptamer may be a good alternative detection probe for the rapid and sensitive detection of SARS.

Investigation of C-terminal domain of SARS nucleocapsid protein–Duplex DNA interaction using transistors and binding-site models

“…Since both dsDNA and RNA have strong binding affinity with the SARS-N protein-CTD, we believe that the values of our dissociation constants are in a reasonable range. In another literature, ssDNA aptamer was reported to have dissociation constant as 4.9 nM when binding to the SARS-N protein-CTD [32]. Although the sequence of the RNA aptamer and the ssDNA aptamer is different from ours, the values of the reported dissociation constants are consistent with our results.…”

Use of Aptamers as Diagnostics Tools and Antiviral Agents for Human Viruses

“…Nucleocapsid (N) protein is the target choice as it is one of the most abundant structural proteins. Both DNA and RNA aptamers against N protein have been developed with Kd of 4.93 and 1.65 nM, respectively [190,191]. DNA aptamer efficiently detects N protein by Western blot suggesting that it could be an alternative to the antibody.…”

Aptasensors for viral diagnostics

“…Recently, a whole range of virus-specific aptamers were generated against vaccinia virus [36,40], dengue virus [41], severe acute respiratory syndrome (SARS) [42][43][44][45], hepatitis C [46][47][48][49][50][51], human immunodeficiency virus (HIV) [52][53][54][55], apple stem pitting virus [56][57][58], bovine viral diarrhea virus [39], norovirus [34,59], rabies virus [60], hepatitis B [61], Ebola [62] and influenza [31,33,35,[63][64][65][66][67][68][69][70][71][72][73][74].…”