DNA is emerging as a robust data storage medium that offers ultrahigh storage densities greatly exceeding conventional magnetic and optical recorders. Information stored in DNA can be copied in a massively parallel manner and selectively retrieved via polymerase chain reaction (PCR). (1−10) However, existing DNA storage systems suffer from high latency caused by the inherently sequential writing process. Despite recent progress, a typical cycle time of solid-phase DNA synthesis is on the order of minutes, which limits the practical applications of this molecular storage platform. (11) Using current technologies, writing 100 bits of information requires nearly 2 h  (11) and costs more than U.S. $1, (12) assuming that each nucleotide stores its theoretical maximum of two bits. To overcome these challenges, new synthesis methods and information encoding approaches are required to accelerate the speed of writing large-volume data sets. (13)

Expanding the alphabet of a DNA storage media by including chemically modified DNA nucleotides can both increase the storage density and the writing speed because more than two bits are recorded during each synthesis cycle. However, designing chemically modified nucleotides as new letters for the DNA storage alphabet must be tightly coupled to the process of reading the encoded information via DNA sequencing, because current DNA sequencing methods, including single-molecule nanopore sequencing, have been developed and optimized to read natural nucleotides. Prior work reported an expanded nucleic acid alphabet of synthetic DNA and RNA nucleotides that can be replicated and transcribed using biological enzymes, (14) but this alphabet was not designed for molecular storage applications and was not accurately read using a nucleic acid sequencing method. Aerolysin nanopores were used to detect synthetic polymers flanked by adenosines, where each monomer of the polymer carries one bit of information. (15) Prior work has reported successful detection of base pairs containing single chemically modified nucleotides (16,17) or discrimination of single nucleotides in natural versus modified states. (18) Despite recent advances, single-molecule detection and sequencing of an expanded molecular alphabet based on a library of chemically diverse modified nucleotides has not yet been demonstrated.

Here, we report an expanded molecular alphabet for DNA data storage comprising four natural and seven chemically modified nucleotides that are readily detected and distinguished using nanopore sequencers (Figure 1 and Table 1). Our results show that Mycobacterium smegmatis porin A (MspA) nanopores, which are widely used for ssDNA sensing and single molecule chemistry studies, (19−21) can accurately discriminate 77 combinations and orderings of chemically diverse monomers within homo- and heterotetrameric sequences (Figures 1, 2, S1, and S2 and Tables S1–S3). We further demonstrate that highly accurate classification (exceeding 60% on average) of combinatorial patterns of natural and chemically modified nucleotides is possible using deep learning architectures that operate on raw current signals generated by GridION of Oxford Nanopore Technologies (ONT) (22) (Figures 3, S3, and S4). We further study the stability of DNA duplexes containing modified nucleotides using all-atom molecular dynamics (MD) simulations (23−26) (Figures 4, S5, and S6 and Table S5). Overall, the extended molecular alphabet has the potential to offer a nearly 2-fold increase in storage density and potentially the same order of reduction in recording latency, thereby providing a promising path forward for the development of new molecular recorders.

Figure 1. DNA data storage using natural and chemically modified nucleotides. (A) Chemical structures of natural DNA nucleotides (A, C, G, T) and the selected chemically modified nucleotides employed in our study (B1–B7). (B) Schematic of the ssDNA oligo used in MspA nanopore experiments. The length of the oligos is 40 nucleotides (nts), with biotin attached at the 5′ terminus. Homo- or heterotetrameric sequences are located at positions 13–16, flanked by two polyT regions of length 12 nt and 24 nt on the 5′ and 3′ ends, respectively. (C) Sequence space for DNA homotetramers or heterotetramers used in MspA nanopore experiments. The notation aX + bY, where a and b take values in {2, 3, 4} so that a + b = 4, indicates that “a” symbols of the same kind are combined with “b” symbols of another kind and arranged in an arbitrary linear order. In total, 77 distinct tetrameric sequences were synthesized and tested experimentally. (Left) Circular diagram showing all 11 homotetramers and 12 tetrameric sequences of the form ACT + X, where X is a chemically modified nucleotide from the set {B2, B3, B5}. (Middle) Circular diagram showing all 30 tested combinations of tetrameric sequences with total composition 2X + 2Y using chemically modified monomers from the set {B1, B2, B3, B4, B5}, including sequence patterns XXYY, XYYX, and XYXY. (Right) Circular diagram showing the remaining 24 combinations of tetrameric sequences with total composition 3X + Y using the set {B2, B3, B5}. Five chemically modified nucleotides form stable base pairs with natural nucleotides via hydrogen bonds (B2–G, B3–A, B5–A, B6–A, B6–C), based on the results from molecular dynamic (MD) simulations.