Deciphering the RNA:DNA triplex binding code of ncRNAs and studying its interaction with chromatin

形成特定的三螺旋，即RNA链与DNA的主要凹槽结合，是长和短非编码RNA序列特异性靶向基因组DNA的一种手段。三螺旋结构被认为在许多核过程中发挥重要作用，如反义介导的基因调控，改变基因组结构域的表观遗传状态，调节染色质密度，基因组可及性，最近有人认为拓扑相关结构域的组织和核结构是由三重rna机制决定的。尽管有许多涉及的功能，但对三螺旋形成的生物物理参数及其与染色质的功能相互作用知之甚少。目前存在三种不同的三螺旋结合基序，但还没有关于序列基序/长度、结合亲和力、突变影响等方面的综合数据，无法预测潜在基序的结合亲和力。我们发现，如果多嘌呤基序在体内作为潜在的结合位点，那么这些基序具有非常不同的和意想不到的结合特性，目前还无法预测。我们证明许多已发表的三联体结合位点预测是错误的。此外，核小体相对于三联体靶向位点的定位强烈影响三联体的稳定性，我们认为组蛋白翻译后修饰将进一步影响结合。我们建议阐明三联体结合密码的生物物理性质，并确定其与染色质的相互作用，以建立预测RNA中潜在三联体形成元件结合亲和力的生物信息学工具。我们认为，我们的工作提供了一个重要的存储库，以解决RNA结合染色质在体内的机械和功能影响。

The formation of specific triple helices, whereby an RNA strand binds to the major groove of DNA, is one means of sequence specific targeting of long and short non-coding RNA to genomic DNA. Triple helix formation is suggested to play an essential role in many nuclear processes, like antisense-mediated gene regulation, changing epigenetic states of genomic domains, regulating chromatin density, genome accessibility and recently it was suggested that the organization of the topologically associated domains and nuclear architecture is determined by a triplex-RNA mechanism. Despite the many implicated functions only little is known about the biophysical parameters of triple helix formation and its functional interaction with chromatin. Three different triple helix binding motifs exist and still no comprehensive data on sequence motifs/lengths, binding affinities, mutational impacts etc. exist, to predict binding affinities of potential motifs. We showed that motifs have very different and unexpected binding properties that currently do not allow to predict, if a poly-purine motif serves as a potential binding site in vivo. We show that many published triplex binding site predictions are wrong. Furthermore, nucleosome positioning relative to the triplex targeting site strongly affects triplex stability and we suggest that the histone post-translational modifications will further affect binding. We propose elucidating the biophysical properties of the triplex binding code and determine its interaction with chromatin to build a bioinformatic tool predicting the binding affinity of potential triplex forming elements in RNA. We think that our work presents an essential repository for addressing the mechanistical and functional impact of RNA binding to chromatin in vivo.