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<p>RNA is an important biomarker for research and diagnostics. However, its transient nature and fragility require its conversion to DNA prior to detection. To this end, enzymatic approaches have been used but are limited by biological constraints, while chemical methods hold much promise but have not been competitive alternatives. Here, we demonstrate that chemical ligation of DNA oligonucleotides hybridised to a complementary RNA template to form an artificial squaramide backbone can be used to quantify sub-attomoles of RNA. This reaction requires mildly buffered, monovalent salt solutions with no extra chemical reagents, and can be performed at a range of temperatures within minutes. We describe the careful design of a three-component ligation system that minimises false-positives and demonstrate its use in qPCR to detect long RNAs in complex systems. Detection limits of 0.3 attomoles are achieved making it one of the most sensitive and specific chemical ligation systems to the best of our knowledge.</p>

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Non-enzymatic conversion of RNA sequence information into DNA by squaramide ligation for accurate RNA quantification

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月19日

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  • Lapatrada Taemaitree,
  • Friedrich Burba,
  • Nina Harvey,
  • Arun Shivalingam,
  • Tom Brown,
  • Afaf H. El-Sagheer

摘要

RNA is an important biomarker for research and diagnostics. However, its transient nature and fragility require its conversion to DNA prior to detection. To this end, enzymatic approaches have been used but are limited by biological constraints, while chemical methods hold much promise but have not been competitive alternatives. Here, we demonstrate that chemical ligation of DNA oligonucleotides hybridised to a complementary RNA template to form an artificial squaramide backbone can be used to quantify sub-attomoles of RNA. This reaction requires mildly buffered, monovalent salt solutions with no extra chemical reagents, and can be performed at a range of temperatures within minutes. We describe the careful design of a three-component ligation system that minimises false-positives and demonstrate its use in qPCR to detect long RNAs in complex systems. Detection limits of 0.3 attomoles are achieved making it one of the most sensitive and specific chemical ligation systems to the best of our knowledge.