Validation of Affinity Differences between RNA Aptamers and a Target Protein using MALDI-TOF MS

Aptamers are oligonucleotides or peptide molecules that bind to a specific target molecule with high affinity. In particular, nucleic acid aptamers can be selected among randomized sequences using the Systematic Evolution of Ligands by Exponential enrichment (SELEX) method, in which selections of oligonucleotides that bind to the target molecule are iterated. Thus, selection of nucleic acid aptamers with a high affinity entails a legitimate assay to differentiate affinities among aptamer candidates. Several affinity assay methods have been adapted to isolate the nucleic acid aptamer that selectively binds to the target protein, including an electrophoretic mobility shift assay, nitrocellulose filter binding assay, and affinity chromatography. For example, the nitrocellulose filter binding assay utilizes a property of the nitrocellulose filter that retains only oligonucleotides that are bound to the protein. Unbound oligonucleotides pass through the nitrocellulose filter without retention onto the filter membrane. However, in general, these methods require moderate amounts of the target protein, as well as a procedure for labeling oligonucleotides for discernment of signals from background reading. In this communication, we report on a method for simple validation of affinity difference between candidate RNA aptamers obtained through screening, thus, facilitating optimum aptamer determination by combination of aptamerpresenting chips and matrix assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF MS). As a model system, RNA aptamers against Severe Acute Respiratory Syndrome (SARS) coronavirus helicase (SCV helicase) were employed. SARS coronavirus, which claimed almost 800 lives in southern China within a period of a few months between 2002 and 2003, is a serious concern worldwide due to absence of a vaccine or effective drug. The SARS viral genome encodes a number of nonstructural proteins (nsPs), which are indispensible for viral RNA genome replication. Among these, nsP13 harboring RNA helicase activity was suggested as an attractive target for development of an anti viral agent. We previously isolated inhibitory RNA aptamer pools that bind to SCV helicase (nsP13), which showed efficient inhibition of viral helicase activity for unwinding duplex DNA. Our strategy utilized self-assembled monolayers (SAMs) on gold to prepare aptamer-presenting chips. Figure 1(a) shows the structure of the monolayer and chemical reactions taking place on the surface. The carboxylic acid-presenting monolayer which was prepared with tri(ethylene glycol)terminated alkanethiol and acid penta(ethylene glycol)terminated alkanethiol in a ratio of 10:90, was coupled to Naminoethyl maleimide in order to introduce thiol groups. Thiolated anchor DNA was then conjugated to the resulting maleimide by way of the Michael addition. Surrounding tri(ethylene glycol) groups provide inertness to nonspecific adsorption of proteins which is the most demanding feature