Rolling Circle Amplification with an Expanded Genetic Alphabet (ExRCA) Generates DNA Materials Incorporating Additional Building Blocks for Enhanced Programmable Functionalization

The development of unnatural base pairs (UBPs) has expanded the genetic alphabet, resulting in a tremendous increase in the information capacity and function diversity of DNA. Efficient amplification of DNA containing UBPs is crucial for the broad use of the UBPs. As one of the major methods for DNA amplification, rolling circle amplification has been extensively employed in many fields ranging from biotechnology to DNA nanotechnology. Herein, we explored and revealed the good efficiency and fidelity of the phi29 DNA polymerase for replicating a representative UBP, dNaM-dTPT3, and its analogues. Based on this, we established the method of rolling circle amplification with an expanded genetic alphabet (ExRCA), together with several strategies for preparing the circular templates for ExRCA. We next demonstrated the application of ExRCA for the mass production or amplification of single- and double-stranded DNAs containing an unnatural base or UBP, as well as the use of the single-stranded DNA product for constructing a site-specifically labeled DNA tetrahedron. The great application potential of ExRCA in biotechnology and materials science of DNA was further demonstrated by utilizing ExRCA to develop an ultrasensitive method for biomolecule detection, DNA hydrogels site-specifically loaded with various cargos, programmable multivalent aptaprobes, a fine-tuned DNA cascade catalyst, and a functional DNA hydrogel with site-specific cross-links. This work not only offers an efficient method for isothermal amplification of DNA containing unnatural nucleobases but also exhibits the unique value of the UBPs with natural-like replication efficiency in constructing DNA materials with increased number of building blocks and programmable functionalities.