Department of Chemistry and Biochemistry, Stern College for Women, Yeshiva University, New York, New York, United States of America.
Department of Chemistry and Biochemistry, Yeshiva College, Yeshiva University, New York, New York, United States of America.
PLoS Comput Biol. 2023 Aug 25;19(8):e1011418. doi: 10.1371/journal.pcbi.1011418. eCollection 2023 Aug.
Nucleic acid probes are valuable tools in biology and chemistry and are indispensable for PCR amplification of DNA, RNA quantification and visualization, and downregulation of gene expression. Recently, triplex-forming oligonucleotides (TFO) have received increased attention due to their improved selectivity and sensitivity in recognizing purine-rich double-stranded RNA regions at physiological pH by incorporating backbone and base modifications. For example, triplex-forming peptide nucleic acid (PNA) oligomers have been used for imaging a structured RNA in cells and inhibiting influenza A replication. Although a handful of programs are available to identify triplex target sites (TTS) in DNA, none are available that find such regions in structured RNAs. Here, we describe TFOFinder, a Python program that facilitates the identification of intramolecular purine-only RNA duplexes that are amenable to forming parallel triple helices (pyrimidine/purine/pyrimidine) and the design of the corresponding TFO(s). We performed genome- and transcriptome-wide analyses of TTS in Drosophila melanogaster and found that only 0.3% (123) of total unique transcripts (35,642) show the potential of forming 12-purine long triplex forming sites that contain at least one guanine. Using minimization algorithms, we predicted the secondary structure(s) of these transcripts, and using TFOFinder, we found that 97 (79%) of the identified 123 transcripts are predicted to fold to form at least one TTS for parallel triple helix formation. The number of transcripts with potential purine TTS increases when the strict search conditions are relaxed by decreasing the length of the probe or by allowing up to two pyrimidine inversions or 1-nucleotide bulge in the target site. These results are encouraging for the use of modified triplex forming probes for live imaging of endogenous structured RNA targets, such as pre-miRNAs, and inhibition of target-specific translation and viral replication.
核酸探针是生物学和化学领域非常有价值的工具，在 DNA 的 PCR 扩增、RNA 定量和可视化以及基因表达下调方面不可或缺。最近，由于在生理 pH 下通过引入骨架和碱基修饰来识别富含嘌呤的双链 RNA 区域，三链形成寡核苷酸 (TFO) 受到了越来越多的关注。例如，三链形成肽核酸 (PNA) 寡聚物已被用于对细胞内结构 RNA 进行成像并抑制流感 A 复制。尽管有一些程序可用于识别 DNA 中的三链靶标 (TTS)，但没有可在结构 RNA 中找到此类区域的程序。在这里，我们描述了 TFOFinder，这是一个 Python 程序，可方便地识别适用于形成平行三螺旋 (嘧啶/嘌呤/嘧啶) 的分子内嘌呤 RNA 双链体，并设计相应的 TFO。我们对黑腹果蝇的基因组和转录组范围内的 TTS 进行了分析，发现只有 0.3%（123）的总独特转录本（35642）显示出形成 12 个嘌呤长三链形成位点的潜力，这些位点至少包含一个鸟嘌呤。使用最小化算法，我们预测了这些转录本的二级结构，并且使用 TFOFinder，我们发现，在所鉴定的 123 个转录本中，有 97%（79%）被预测为折叠形成至少一个 TTS，以用于平行三螺旋形成。当通过减少探针长度或允许靶标中最多两个嘧啶反转或 1 个核苷酸凸起来放宽严格的搜索条件时，具有潜在嘌呤 TTS 的转录本数量会增加。这些结果对于使用修饰的三链形成探针对内源结构 RNA 靶标（如 pre-miRNA）进行实时成像以及抑制靶标特异性翻译和病毒复制是令人鼓舞的。
