Fluorescent probes for sensing nucleic acids have found widespread use in the field of cell and molecular biology. However, probes combined with potential for post-synthetic conjugation, e.g. for intra-endosomal measurements of RNA, are unavailable. Herein we developed cyanine dyes that can be conjugated to viral capsid or other targets. First, we solved the crystal structure of SYBR Green II. The structural elucidation of this commonly used RNA probe provided the basis for synthesizing similar molecules with much desired function for post-synthetic conjugation. To address this need, cyanine dyes were prepared using an alternative synthesis protocol. All studied compounds showed considerable brightness upon binding to nucleic acids. However, regardless of the common chromophore on the dyes, the observed fluorescence emission intensities varied significantly, where methyl-substituted dye 1 gave values higher than SYBR Green II, whereas compounds 2–5 containing undecyl spacers had lower values. Studying the structure-activity relationship revealed the longer alkyl chains to induce slight perturbation in dye intercalation, as well as demanding larger binding area on the nucleic acid lattice, explaining these differences. To study the potential biological use of the dyes, the RNA genome of enterovirus echovirus 1 was studied in vitro with the probes. A novel method employing the low binding space requirement of 1 was developed to determine the single-to-double-stranded RNA ratio of a sample, whereas compound 4 was covalently bound to the viral capsid and used successfully to monitor the viral RNA release from within the capsid. The presented results open new possibilities for preparation and use of SYBR Green-based nucleic acid probes to further apply these compounds for increasingly demanding targeting in biological contexts.

已经发现用于感测核酸的荧光探针在细胞和分子生物学领域中得到了广泛的应用。但是，探针与合成后结合的潜力结合在一起，例如无法用于RNA的内膜内测量。在本文中，我们开发了可以与病毒衣壳或其他靶标结合的花菁染料。首先，我们解决了SYBR Green II的晶体结构。该常用RNA探针的结构阐明为合成具有合成后缀合所需功能的相似分子提供了基础。为了满足该需求，使用替代合成方案制备了花青染料。所有研究的化合物与核酸结合后均显示出相当大的亮度。但是，不管染料上的常见生色团如何，观察到的荧光发射强度都发生了显着变化，其中甲基取代的染料1的值高于SYBR Green II，而化合物2 – 5包含十一烷基间隔基的值较低。对结构活性关系的研究表明，更长的烷基链会在染料嵌入中引起轻微的扰动，并要求在核酸晶格上具有更大的结合面积，从而解释了这些差异。为了研究这种染料的潜在生物学用途，用探针在体外研究了肠病毒回声病毒1的RNA基因组。采用低的结合的空间需求的新方法1的开发是为了确定单到双链的样品的RNA比，而化合物4病毒与衣壳共价结合，并成功地用于监测衣壳内部病毒RNA的释放。提出的结果为基于SYBR Green的核酸探针的制备和使用开辟了新的可能性，以进一步将这些化合物应用于生物学环境中日益严格的靶向。