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

Aptamer sequences obtained from the selected clones.

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