Isolation of inhibitory RNA aptamers against severe acute respiratory syndrome (SARS) coronavirus NTPase/Helicase

Abstract

Recent outbreak of Severe Acute Respiratory Syndrome (SARS) that caused almost 800 victims requires a development of efficient inhibitor against SARS coronavirus (SCV). In this study, RNA aptamers against SCV NTPase/Helicase (nsP10) were isolated from RNA library containing random sequences of 40 nts using in vitro selection technique. Nucleotide sequences of enriched RNA aptamer pool (ES15 RNA) contain AG-rich conserved sequence of 10-11 nucleotides [AAAGGR(G)GAAG; R, purine base] and/or additional sequence of 5 nucleotides [GAAAG], which mainly reside at the loop region in all the predicted secondary structures. Isolated RNAs were observed to efficiently inhibit double-stranded DNA unwinding activity of the helicase by up to approximately 85% with an IC(50) value of 1.2nM but show a slight effect on ATPase activity of the protein in the presence of cofactor, poly (rU). These results suggest that the pool of selected aptamers might be potentially useful as anti-SCV agents.

Fig. 1

The sequence of RNA pool for in vitro selection and selected RNA aptamers against SCV nsP10. (A) The RNA library was produced by in vitro transcription of the DNA template containing 40 random nucleotides. (B) The 6 different RNA sequences identified in the ES15 RNA pool are shown. These RNAs of three groups were identified to contain a AG-rich conserved sequence of 10–11 nucleotides (in white boxes) in the middle of core region. Group II and III are observed to include an additional conserved sequence (in gray boxes).

Fig. 2

Secondary structures of RNA aptamers derived from the ES15 RNA pool. The secondary structures of 6 different RNA sequences identified in the ES15 RNA pool were predicted using the MFold program. The conserved motifs (gray-colored sequences) mostly reside at the loop region.

Fig. 3

Effects of ES15 RNA pool on the ATPase activity of SCV NTPase/Helicase. (A) ATP hydrolysis reaction by the protein was performed in the presence of either initial random RNA pool (○) or ES15 RNA pool (●). ATP hydrolysis reaction was carried out as described in “Materials and methods”. Hyperbolic fit (solid line) of the ES15 RNA-stimulation profile provided K_1/2 value of 2.34 ng/μl for RNA-stimulated ATP hydrolysis by the SCV NTPase/Helicase. (B) Poly (rU) (100 nM)-stimulated ATP hydrolysis reaction by the protein was performed in the presence of either initial random RNA pool (○) or ES15 RNA pool (●). Values shown are averages of measurements performed in triplicate (graphs in panel A and B). (C) Averaged molecular weight of ES15 RNA pool was determined to be 30.2 kDa by MALDI TOF-MS.

Fig. 4

Principles of the FRET-based helicase assay and inhibition of the SCV helicase activity by the ES15 RNA. (A) Schematic drawing of the FRET-based dsDNA unwinding assay of helicase activity. Each T20D25 and T0D25 single stranded DNA labeled with TAMRA and Fluorescein on 3′-end and 5′-end, respectively, were annealed and then incubated at 37 °C or 95 °C (heat denaturation) in the absence or presence of SCV helicase for 10 min. The unwound DNA by heat treatment (■) emitted higher fluorescence than other cases. (B) Inhibition of the helicase activity of the SCV helicase in the presence of various concentrations of the ES15 RNA. Data of the ES15 RNA (○) were fitted to the hyperbolic equation (solid line), which provide an IC₅₀ of 0.035 ng/μl (equivalent to 1.2 nM) and the extent of maximal inhibition (85%). For a comparison, random RNA pool (●) was also tested for the same dsDNA unwinding assay. Points shown in graph are the average of triplicate experiments performed separately.