ABSTRACT
Natural DNA is exquisitely evolved to store genetic information. The chirally inverted L-DNA, possessing the same informational capacity but resistant to biodegradation, may serve as a robust, bioorthogonal information repository. Here we chemically synthesize a 90-kDa high-fidelity mirror-image Pfu DNA polymerase that enables accurate assembly of a kilobase-sized mirror-image gene. We use the polymerase to encode in L-DNA an 1860 paragraph by Louis Pasteur that first proposed a mirror-image world of biology. We realize chiral steganography by embedding a chimeric D-DNA/L-DNA key molecule in a D-DNA storage library, which conveys a false or secret message depending on the chirality of reading. Furthermore, we show that a trace amount of an L-DNA barcode preserved in water from a local pond remains amplifiable and sequenceable for 1 year, whereas a D-DNA barcode under the same conditions could not be amplified after 1 day. These next-generation mirror-image molecular tools may transform the development of advanced mirror-image biology systems and pave the way for the realization of the mirror-image central dogma and exploration of their applications.
Subject(s)
DNA-Directed DNA Polymerase , DNA , DNA/chemistry , DNA/genetics , Information Storage and Retrieval , StereoisomerismABSTRACT
After realizing mirror-image genetic replication, transcription, and reverse transcription, the biggest challenge in establishing a mirror-image version of the central dogma is to build a mirror-image ribosome-based translation machine. Here, we chemically synthesized the natural and mirror-image versions of three ribosomal proteins (L5, L18, and L25) in the large subunit of the Escherichia coli ribosome with post-translational modifications. We show that the synthetic mirror-image proteins can fold inâ vitro despite limited efficiency and assemble with enzymatically transcribed mirror-image 5S ribosomal RNA into ribonucleoprotein complexes. In addition, the RNA-protein interactions are chiral-specific in that the mirror-image ribosomal proteins do not bind with natural 5S ribosomal RNA and vice versa. The synthesis and assembly of mirror-image 5S ribonucleoprotein complexes are important steps towards building a functional mirror-image ribosome.
Subject(s)
RNA, Ribosomal, 5S/metabolism , Ribosomal Proteins/metabolism , Amino Acid Sequence , Circular Dichroism , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Protein Binding , RNA, Ribosomal, 5S/chemistry , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Ribosomal Proteins/chemical synthesis , Ribosomal Proteins/chemistry , Ribosomal Proteins/genetics , Ribosomes/metabolism , StereoisomerismABSTRACT
The construction of mirror-image biological systems may open the next frontier for biomedical technology development and discovery. Here we have designed and chemically synthesized a mutant version of the thermostable Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) consisting of d-amino acids. With a total peptide length of 358 amino acid residues, it is the largest chemically synthesized d-amino acid protein reported to date. We show that the d-polymerase is able to amplify a 120-bp l-DNA sequence coding for the Escherichia coli 5S ribosomal RNA gene rrfB by mirror-image polymerase chain reaction, and that both the natural and mirror-image systems operate with strict chiral specificity. The development of efficient miPCR systems may lead to many practical applications, such as mirror-image systematic evolution of ligands by exponential enrichment for the selection of therapeutically promising nuclease-resistant l-nucleic acid aptamers.
