Grupo de Aptámeros. Departamento de Bioquímica-Investigación, Hospital Universitario Ramón y Cajal (IRYCIS), Carretera de Colmenar Viejo Km.9.100, CP-28034, Madrid, Spain.
Grupo de Aptámeros. Departamento de Bioquímica-Investigación, Hospital Universitario Ramón y Cajal (IRYCIS), Carretera de Colmenar Viejo Km.9.100, CP-28034, Madrid, Spain; Aptus Biotech SL, Av. Cardenal Herrera Oria 298, CP-28035, Madrid, Spain.
Anal Chim Acta. 2020 Apr 22;1107:155-163. doi: 10.1016/j.aca.2020.02.012. Epub 2020 Feb 7.
Leishmaniasis is a disease caused by a parasite of the genus Leishmania that affects millions of people worldwide. These parasites are characterized by the presence of a DNA-containing granule, the kinetoplastid, located in the single mitochondrion at the base of the cell's flagellum. Interestingly, these flagellates do not condense chromatin during mitosis, possibly due to the specific molecular features of their histones. Although histones are extremely conserved proteins, kinetoplastid core histone sequences diverge significantly from those of higher eukaryotes. This divergence makes kinetoplastid core histones potential diagnostic and/or therapeutic targets. Aptamers are short single-stranded nucleic acids that are able to recognize target molecules with high affinity and specificity. Their binding capacity is a consequence of the particular three-dimensional structure acquired depending on their sequence. These molecules are currently used for detection, diagnosis and therapeutic purpose. Starting from a previously obtained ssDNA aptamer population against rLiH3 protein we have isolated two individual aptamers, AptLiH3#4 and AptLiH3#10. Next, we have performed ELONA, Western blot and slot blot assays to establish aptamer specificity and affinity for LiH3 histone. In addition, ELONA assays using peptides corresponding to overlapped sequences of LiH3 were made to map the aptamers:LiH3 interaction. Finally, different assays using aptamers were performed in order to evaluate the possibility of using these aptamers as sensing molecule to recognize the endogenous protein LiH3. Our results indicate that both aptamers have high affinity and specificity for the target and are able to detect the endogenous LiH3 histone protein in promastigotes lysates. In silico analysis reveals that these two aptamers have different potential secondary structure among them, however, both of them are able to recognize the same peptide sequences present in the protein. In conclusion, our findings indicate that these aptamers could be used for LiH3 histone detection and, in consequence, as potential biosensing molecules in a diagnostic tool for leishmaniasis.
利什曼病是一种由利什曼原虫属寄生虫引起的疾病，影响着全球数百万人。这些寄生虫的特征是存在一个含有 DNA 的颗粒，即动基体，位于细胞鞭毛基部的单个线粒体中。有趣的是，这些鞭毛在有丝分裂过程中不会浓缩染色质，这可能是由于它们组蛋白的特定分子特征所致。尽管组蛋白是高度保守的蛋白质，但动基体核心组蛋白序列与高等真核生物的序列有很大的差异。这种差异使得动基体核心组蛋白成为潜在的诊断和/或治疗靶点。适配体是能够与靶分子高亲和力和特异性结合的短单链核酸。它们的结合能力是由于其序列决定的特定三维结构获得的。这些分子目前用于检测、诊断和治疗目的。从之前获得的针对 rLiH3 蛋白的 ssDNA 适配体群体中，我们分离出了两个单独的适配体，AptLiH3#4 和 AptLiH3#10。接下来，我们进行了 ELISA、Western blot 和 slot blot 检测，以确定适配体对 LiH3 组蛋白的特异性和亲和力。此外，还进行了 ELONA 检测，使用与 LiH3 重叠序列对应的肽段来绘制适配体与 LiH3 的相互作用图谱。最后，还进行了不同的适配体检测实验，以评估将这些适配体用作识别内源性 LiH3 蛋白的传感分子的可能性。我们的研究结果表明，这两种适配体对靶标均具有高亲和力和特异性，并且能够检测到前鞭毛体裂解物中的内源性 LiH3 组蛋白蛋白。计算机分析表明，这两种适配体之间存在不同的潜在二级结构，但它们都能够识别存在于该蛋白中的相同肽段序列。总之，我们的研究结果表明，这些适配体可用于 LiH3 组蛋白的检测，因此可作为利什曼病诊断工具中的潜在生物传感分子。
