Aptamers as biomarkers for neurological disorders. Proof of concept in transgenic mice.

The act of selecting aptamers against blood serum leads to deep libraries of oligonucleotide sequences that bind to a range of epitopes in blood. In this study we developed an enriched aptamer library by performing positive selection against a pool of blood serum samples from transgenic mice (P301S) carrying the human tau gene and counter selecting against pooled blood serum from negative segregant (wild type) mice. We demonstrated that a large proportion of the aptamer sequences observed with next generation sequence (NGS) analysis were the same from selection round 5 and selection round 6. As a second step, we applied aliquots of the selection round 5 enriched library to blood serum from 16 individual mice for a single round of selection. Each of these individual libraries were characterized through NGS analysis and the changes in relative frequency in aptamers from transgenic mice versus wild type were used to construct a diagnostic fingerprint of the effect of the action of the transgene on the composition of blood serum. This study serves as a model for similar applications with human subjects

Phylogram of correlation among 1000 aptamers across 16 mice.

<p>Relative distances correlations for aptamer frequencies across the 16 mice. Distances are Euclidean distances between r² values from the correlation matrix, and the hierarchal clustering was performed using the ward.D2 algorithm.</p

Individual comparison between transgenic and wild type mice.

<p>The X axis represents each individual mouse as noted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190212#pone.0190212.t001" target="_blank">Table 1</a>. Y axis provides the Aptamarker score according to the algorithm.</p

Sequence enrichment for the unselected library and selection round 5 and 6.

<p>On the x axis are the frequencies of each aptamer sequence observed by NGS analysis. On the y axis are the frequencies of the observation of these frequencies. SR5 = selection round 5, SR6 = selection round 6. Unselected = Purchased library from Trilink Biotechnologies.</p

Overall comparison between transgenic and wild type mice.

<p>The averages across the transgenic and the wt mice in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190212#pone.0190212.g009" target="_blank">Fig 9</a> were used to generate the data presented in this figure.</p

Experimental design.

<p>Experimental design.</p

Unrooted phylogram of 1,000 aptamers across 16 mice defining clusters.

<p>Unrooted phylogram of 1,000 aptamers across 16 mice defining clusters.</p

Mice used for individual serum selection.

<p>Mice used for individual serum selection.</p

Analysis of randomly generated aptamer frequency data.

<p>The distance matrix based on randomly generated aptamer frequencies with the same mean and standard deviation as the observed aptamer frequencies plotted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190212#pone.0190212.g006" target="_blank">Fig 6</a>. Analytical approach was identical to that described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190212#pone.0190212.g006" target="_blank">Fig 6</a>.</p

Correlation heat map of 1,000 aptamers across 16 mice.

<p>Based on the same distance matrix used for Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190212#pone.0190212.g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190212#pone.0190212.g005" target="_blank">5</a>, and on the ward.D2 algorithm for hierarchal clustering. Relative distances between aptamer frequencies are plotted in a colour gradient with dark blue being equal to +1, and dark red being equal to -1. Each row and each column correspond to a different specific aptamer.</p