Reading mixtures of uniform sequence-defined macromolecules to increase data storage capacity

Abstract

In recent years, the field of molecular data storage has emerged from a niche to a vibrant research topic. Herein, we describe a simultaneous and automated read-out of data stored in mixtures of sequence-defined oligomers. Therefore, twelve different sequence-defined tetramers and three hexamers with different mass markers and side chains are successfully synthesised via iterative Passerini three-component reactions and subsequent deprotection steps. By programming a straightforward python script for ESI-MS/MS analysis, it is possible to automatically sequence and thus read-out the information stored in these oligomers within one second. Most importantly, we demonstrate that the use of mass-markers as starting compounds eases MS/MS data interpretation and furthermore allows the unambiguous reading of sequences of mixtures of sequence-defined oligomers. Thus, high data storage capacity considering the field of synthetic macromolecules (up to 64.5 bit in our examples) can be obtained without the need of synthesizing long sequences, but by mixing and simultaneously analysing shorter sequence-defined oligomers.

Fig. 1. Concept of the automated read-out of a mixture of sequence-defined molecules by varying 12 different aldehydes and specifically designed mass markers (TAGs).

Iterative step synthesis with the Passerini three component reaction (P-3CR), using twelve different aldehydes and three different TAGs. The aldehydes can be introduced at any desired position of the oligomer and provide the sidechains of the macromolecule and thus differentiate each repeating unit. Subsequently, the individual sequences of an oligomer mixture can be analyzed via ESI-MS and ESI-MS/MS, followed by fully automated read-out with the computer program with a clearly defined position of the TAGs.

Fig. 2. Schematic representation of the variation of the twelve different aldehydes (colored bullets) and SEC traces of three different tetramers, one for each tag.

a Chemical structure and SEC traces of a tetramer with TAG1 and the sidechain variation of the aldehydes 14a–l (see Supplementary Methods for detailed information) for another three tetramers. b Chemical structure and SEC traces of a tetramer with TAG2 and the sidechain variation of the aldehydes 14a–l (see Supplementary Methods for detailed information) for another three tetramers. c Chemical structure and SEC traces of a tetramer with TAG3 and the sidechain variation of the aldehydes 14a–l (see Supplementary Methods for detailed information) for another three tetramers. d Chemical structures of TAG1–3.

Fig. 3. Characterization of the sequence-defined hexamer with TAG1.

a Chemical structure of the sequence-defined hexamer H2. b SEC traces of each P-3CR product. c High-resolution of the ESI-MS measurement of H2; calculated isotopic pattern (red) and measured isotopic pattern (black).

Fig. 4. Most common fragmentation patterns of the oligomer during fragmentation by tandem ESI-MS/MS.

a Fragmentation next to the carbonyl, which is preferred in measurements without any additives. b The fragmentation next to the ester group is preferred when sodium trifluoroacetate is used as additive.

Fig. 5. Read-out of the sequence-defined hexamer H2.

Read-out of the hexamer H2 via tandem ESI-MS/MS with an NCE of 18. In the spectrum the read-out from both ends of the oligomer is shown, using the fragmentation next to the carbonyl.

Fig. 6. Read-out of a mixture of three hexamers, with clearly defined positions of the TAGs to increase the data storage capacity.

a ESI-MS spectrum of a mixture of three different hexamers H1-H3 that was used for subsequent tandem ESI-MS/MS fragmentation. For the fragmentation, one of the respective molecule peaks was chosen at a time. b fragmentation of hexamer H1. c fragmentation of hexamer H2. d fragmentation of hexamer H3.