Dual sequence definition increases the data storage capacity of sequence-defined macromolecules

Abstract

Sequence-defined macromolecules offer applications in the field of data storage. Challenges include synthesising precise and pure sequences, reading stored information and increasing data storage capacity. Herein, the synthesis of dual sequence-defined oligomers and their application for data storage is demonstrated. While applying the well-established Passerini three-component reaction, the degree of definition of the prepared monodisperse macromolecules is improved compared to previous reports by utilising nine specifically designed isocyanide monomers to introduce backbone definition. The monomers are combined with various aldehyde components to synthesise dual-sequence defined oligomers. Thus, the side chains and the backbones of these macromolecules can be varied independently, exhibiting increased molecular diversity and hence data storage capacity per repeat unit. In case of a dual sequence-defined pentamer, 33 bits are achieved in a single molecule. The oligomers are obtained in multigram scale and excellent purity. Sequential read-out by tandem ESI-MS/MS verifies the high data storage capacity of the prepared oligomers per repeat unit in comparison to other sequence defined macromolecules.

Fig. 1. Synthesis strategy for dual sequence-defined oligomers.

The two-step iterative cycle allows for independent variation of side chain and backbone. The set of selectable side chains and backbone moieties is depicted in Supplementary Fig. 77.

Fig. 2. Monomers M1–M9, prepared from the corresponding amino acids.

M1–M8 are prepared in three steps; *) in case of M9, the synthesis was performed via a four-step procedure. By variation of these monomers in the iterative synthesis cycle depicted in Fig. 1, different backbone moieties can be introduced to the macromolecules, thus leading to a backbone-defined sequence.

Fig. 3. Three-step synthesis procedure of monomer M2.

The synthesis starts with the esterification of the amino acid, followed by the N-formylation and a final dehydration step. The procedure was applied in the synthesis of monomers M1–M8.

Fig. 4. Characterisation of the backbone-defined heptamer B7.

a Chemical structure of B7. b SEC results after each P-3CR reaction of the iterative synthesis cycle, verifying the high purity of the products. The colour code identifies the respectively used backbone moieties (Fig. 4a). c High-resolution ESI-MS measurement: calculated and observed isotopic pattern of singly protonated product B7. See Supplementary Methods for further characterisation of the products after each reaction.

Fig. 5. Characterisation of the dual sequence-defined pentamer DS5.

a Chemical structure of DS5. b SEC results after each P-3CR reaction of the iterative synthesis cycle, verifying the high purity of the products. The colour code identifies the respectively used side-chains and backbone moieties (Fig. 5A). c High-resolution ESI-MS measurement: calculated and observed isotopic pattern of singly protonated product DS5. The corresponding sodium ion was also found (see Supplementary Methods, also for further characterisation of the products after each reaction).

Fig. 6. Sequential read-out of the dual sequence-defined pentamer DS5.

Read-out is achieved via fragmentation by tandem mass spectrometry, revealing the expected fragmentation pattern. In the spectrum, the fragmentation next to the carbonyl group from both ends of the oligomer is depicted. By recombining the fragments, the initial structure of the pentamer can be re-established and thus the stored information is read. Assignment of the middle fragments as well as the second fragmentation pattern are provided in Supplementary Figs. 135–137.

Fig. 7. Comparison of different data storage systems.

Artificial (i.e. digital, binary) and naturally (i.e. DNA, quaternary) applied data storage systems are compared with the herein discussed macromolecular data storage systems. The number of permutations of five tetramers are depicted. Calculations are based on ^a11 possible side chains, demonstrated herein; ^b9 monomers introduced herein; ^cthe combination of these 11 possible side chains and 9 possible monomers. Please note the logarithmic scale.