Squaramides and Ureas: A Flexible Approach to Polymerase‐Compatible Nucleic Acid Assembly

• Published in: Angewandte Chemie International Edition 2020-07, Vol.59 (28), p.11416-11422
• Main Authors: Shivalingam, Arun, Taemaitree, Lapatrada, El‐Sagheer, Afaf H., Brown, Tom
• Format: Article
• Language: English
• Subjects: Denaturing Gradient Gel Electrophoresis; Deoxyribonucleic acid; DNA; DNA-Directed DNA Polymerase - chemistry; Information retrieval; ligation; Mass Spectrometry; Nucleic acids; Nucleic Acids - chemistry; Oligonucleotides; polymerase chain reaction; Quinine - analogs & derivatives; Quinine - chemistry; Reagents; Reverse transcription; Ribonucleic acid; RNA; RNA detection; squaramide; Urea; Urea - chemistry; Ureas
• ISSN: 1433-7851; 1521-3773
• DOI: 10.1002/anie.202000209

Joining oligonucleotides together (ligation) is a powerful means of retrieving information from the nanoscale. To recover this information, the linkages created must be compatible with polymerases. However, enzymatic ligation is restrictive and current chemical ligation methods lack flexibility. Herein, a versatile ligation platform based on the formation of urea and squaramide artificial backbones from minimally modified 3′‐ and 5′‐amino oligonucleotides is described. One‐pot ligation gives a urea linkage with excellent read‐through speed, or a squaramide linkage that is read‐through under selective conditions. The squaramide linkage can be broken and reformed on demand, while stable pre‐activated precursor oligonucleotides expand the scope of the ligation reaction to reagent‐free, mild conditions. The utility of our system is demonstrated by replacing the enzymatically biased RNA‐to‐DNA reverse transcription step of RT‐qPCR with a rapid nucleic‐acid‐template‐dependent DNA chemical ligation system, that allows direct RNA detection. 3′‐ and 5′‐amino oligonucleotides are chemically ligated through the formation of urea and squaramide artificial backbones. The squaramide linkage can be formed in mild reagent‐free buffered conditions, read‐through accurately by specific polymerases, and even cleaved and reformed on demand. To demonstrate its utility, the RNA‐to‐DNA reverse transcription step of RT‐qPCR is replaced with squaramide chemical ligation for direct RNA detection.