Rapid One-Step Selection Method for Generating Nucleic Acid Aptamers: Development of a DNA Aptamer against alpha-Bungarotoxin

Background: Nucleic acids based therapeutic approaches have gained significant interest in recent years towards the development of therapeutics against many diseases. Recently, research on aptamers led to the marketing of Macugen (R), an inhibitor of vascular endothelial growth factor (VEGF) for the treatment of age related macular degeneration (AMD). Aptamer technology may prove useful as a therapeutic alternative against an array of human maladies. Considering the increased interest in aptamer technology globally that rival antibody mediated therapeutic approaches, a simplified selection, possibly in one-step, technique is required for developing aptamers in limited time period

Aptamer sequences obtained from the selected clones.

<p>Only the sequences from the random regions are shown.</p

Aptamer characterization.

<p>A. Binding specificity of the aptamer obtained from clone 24 & 51 by surface plasmon resonance (SPR) technique using Biacore 3000. Various concentrations of α-bungarotoxin (1, 10, 20, 30, and 40 µM) were passed through the aptamer which was immobilized on a streptavidin coated sensor chip. The obtained sensorgrams demonstrate the aptamer binding to α-bungarotoxin; B. Predicted structure of the obtained aptamer using the DNA folding platform from mfold web server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041702#pone.0041702-Berezovski1" target="_blank">[26]</a>.</p

PCR amplification using the cDNA template generated by reverse transcription by SuperScript® III Reverse Transcriptase.

<p>(A) Primer and template sequences; (B) Agarose gel electrophoresis image of PCR using the cDNA template containing LNA-T generated by SuperScript® III Reverse Transcriptase, lane 1: DNA size markers in base-pairs, lane 2: PCR using the cDNA with natural DNA nucleotides, lane 3: PCR using cDNA template with LNA-T (43 nt), lane 4: PCR without using a template (negative control); (C) Agarose gel electrophoresis image of PCR using the cDNA template with natural DNA nucleotides and LNA-T nucleotides generated from an LNA-modified RNA template T2 by SuperScript® III Reverse Transcriptase, lane 1: DNA size markers in base-pairs, lane 2: PCR using the cDNA with natural DNA nucleotides, lane 3: PCR using cDNA template with LNA-T, lane 4: PCR without using a template (negative control); (D) PCR product analysis by cloning and sequencing, PCR product obtained from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035990#pone-0035990-g004" target="_blank">Figure 4C</a> (lane 2) aligned with the sequencing chromatogram. PCR product containing DNA-T and DNA-A at the expected positions of the LNA nucleotides of the initial RNA template is matched with a double headed arrow as identified in the sequencing chromatogram.</p

Structural representations of LNA monomers.

<p>Structural representations of LNA monomers.</p

Enzymatic incorporation of LNA-T, LNA-A, dT/LNA-T and dA nucleotides using RNA and LNA/RNA templates.

<p>(A) Primer and template sequences. (B) Gel electrophoresis of LNA-T and LNA-A incorporation on a natural RNA template T1, lane 1: Primer, 19 nt; lane 2: Positive control using all four natural dNTPs; lane 3: Negative control reaction with only two natural nucleotides (dCTP, dGTP); lane 4: Incorporation of LNA-T and LNA-A (dCTP, dGTP, LNA-ATP, LNA-TTP in the mixture). (C) Gel electrophoresis of dT/LNA-T and dA nucleotide incorporation using a LNA-A and LNA-T-modified RNA template T2, lane 1: Primer, 19 nt; lane 2: Positive control using all four natural dNTPs; lane 3: Negative control reaction with only three natural nucleotides (dCTP, dATP, dGTP); lane 4: Incorporation of LNA-T (dCTP, dATP, dGTP, LNA-TTP in the mixture). LNA nucleotides are underlined and the incorporation sites in the template RNA are italicized.</p

Enzymatic incorporation of LNA-T nucleotides by SuperScript® III Reverse Transcriptase.

<p>(A) Primer and template sequences. (B) Gel electrophoresis of incorporation LNA-T, lane 1: Primer, 19 nt; lane 2: Positive control using all four natural dNTPs; lane 3: Negative control reaction with only three natural nucleotides (dCTP, dATP, dGTP); lane 4: Incorporation of LNA-T (dCTP, dATP, dGTP, LNA-TTP in the mixture). (C) Gel electrophoresis of incorporation LNA-T at 5–45 minute incubation time points, lane P1: Primer. LNA nucleotides are underlined and the incorporation sites in the template RNA are italicized.</p