Isolation of Single-Stranded DNA Aptamers That Distinguish Influenza Virus Hemagglutinin Subtype H1 from H5

<div><p>Surface protein hemagglutinin (HA) mediates the binding of influenza virus to host cell receptors containing sialic acid, facilitating the entry of the virus into host cells. Therefore, the HA protein is regarded as a suitable target for the development of influenza virus detection devices. In this study, we isolated single-stranded DNA (ssDNA) aptamers binding to the HA1 subunit of subtype H1 (H1-HA1), but not to the HA1 subunit of subtype H5 (H5-HA1), using a counter-systematic evolution of ligands by exponential enrichment (counter-SELEX) procedure. Enzyme-linked immunosorbent assay and surface plasmon resonance studies showed that the selected aptamers bind tightly to H1-HA1 with dissociation constants in the nanomolar range. Western blot analysis demonstrated that the aptamers were binding to H1-HA1 in a concentration-dependent manner, yet were not binding to H5-HA1. Interestingly, the selected aptamers contained G-rich sequences in the central random nucleotides region. Further biophysical analysis showed that the G-rich sequences formed a G-quadruplex structure, which is a distinctive structure compared to the starting ssDNA library. Using flow cytometry analysis, we found that the aptamers did not bind to the receptor-binding site of H1-HA1. These results indicate that the selected aptamers that distinguish H1-HA1 from H5-HA1 can be developed as unique probes for the detection of the H1 subtype of influenza virus.</p></div

Western blot analysis using aptamers.

<p>Various amounts of GST-H1-HA1, GST-H5-HA1, and GST proteins were separated by SDS-PAGE, incubated with 5′-biotinylated aptamers, detected by streptavidin-HRP and ECL visualization (−, 0 μg; +, 1 μg; ++, 5 μg; +++, 10 μg).</p

Binding analysis of selected ssDNA aptamers by ELISA.

<p>(A) Affinity measurements of selected ssDNA aptamers to H1-HA1 by ELISA. Immobilized biotinylated ssDNA aptamers were incubated with increasing concentrations of GST-H1-HA1, and binding was detected with anti-GST antibody-HRP. The biotinylated ssDNA library was used as negative control (◊). Graphs were fitted to the Michaelis-Menten equation, and K_d values were calculated as 64.76 ± 18.24 nM for ApI (●), 69.06 ± 12.34 nM for ApII (△), and 50.32 ± 14.07 nM for ApIII (■). (B) 100 nM GST-H1-HA1, GST-H5-HA1, or GST proteins incubated with biotinylated aptamers immobilized on streptavidin-coated plates to compare binding affinities.</p

G-rich sequence of the core region and CD spectra of selected ssDNA aptamers.

<p>(A) G-rich sequences participating in the formation of G-quadruplex structures as predicted by QGRS Mapper in bold type and underlined. (B) CD spectra as collected on a Chirascan-plus CD spectrometer at 200–320 nm.</p

Flow cytometry analysis.

<p>HEK 293T cells incubated with aptamer-protein complexes containing GST-H1-HA1 or GST-H5-HA1 as analyzed by flow cytometry. The distributions of proteins (column) and selected aptamers (row) are shown.</p

Affinity measurements of biotinylated ssDNA aptamers by SPR.

<p>(A) Biotinylated aptamers immobilized on an NLC sensor chip and interacting with various amounts of GST-H1-HA1. Equilibrium dissociation constants K_D were calculated from association and dissociation rate constants (K_D = k_d/k_a) as 96.6 nM for ApI, 1.09 μM for ApII, and 293 nM for ApIII. (B) Interaction of 10 μM H1-HA1, H5-HA1, or GST with aptamers immobilized on an NLC sensor chip, as measured by SPR.</p

<i>In vitro</i> selection of ssDNA aptamers and specific binding activity of the ssDNA pool.

<p>(A) Schematic representation of the counter-SELEX procedure. After removing ssDNA species binding nonspecifically to glutathione agarose beads, the ssDNA pool was incubated with H5-HA1 (negative control) and centrifuged to remove H5-HA1-binding ssDNAs, after which the unbound ssDNAs were incubated with H1-HA1 (target protein), and the H1-HA1-bound DNAs were extracted using phenol-chloroform and amplified by PCR. After 14 rounds of selection, the enriched DNA was PCR-amplified, cloned, and sequenced. (B) Specific binding activity as measured by ELISA after 8, 10, 12, and 14 rounds of selection. GST-tagged H1-HA1 (100 nM) incubated on selected DNA-coated plates and analyzed using anti-GST antibody-HRP with TMB color detection.</p

Predicted structures and sequence alignments of selected aptamers.

<p>(A) Mfold-predicted secondary structures of selected aptamers with shaded nucleotides representing the 45-nucleotide random sequence region. (B) Conserved sequences identified by ClustalW2 indicated by asterisks and underlines, and shown in (A) as solid lines.</p

Increased susceptibility to Aβ42-mediated cell death in LRP1 minireceptor-expressing cells.

<p>N2a-pcDNA3 and N2a-mLRP4 cells were incubated with increasing concentrations of Aβ42 for 24, 48 and 72 h and cell viability was assessed by the reduction of the MTS dye. Decreased viability was detected only after 72 h incubation with a high concentration of Aβ42 in LRP1 minireceptor expressing cells. * p<0.05, <i>t</i>-test. n = 3.</p

Characterization of a Brain Permeant Fluorescent Molecule and Visualization of Aβ Parenchymal Plaques, Using Real-Time Multiphoton Imaging in Transgenic Mice

Emerging paradigms mandate discovery of imaging agents for diagnosing Alzheimer’s disease (AD) prior to appearance of clinical symptoms. To accomplish this objective, a novel heterocyclic molecule (<b>4</b>) was synthesized and validated as Aβ targeted probe. The agent shows labeling of numerous diffuse Aβ plaques in confirmed AD human brain tissues and traverses the blood–brain barrier to enable labeling of parenchymal Aβ plaques in live mice (APP^±/PS1^±) brains