Controlling the structure of sequence‐defined poly(phosphodiester)s for optimal MS/MS reading of digital information

Abstract

Digital polymers are monodisperse chains with a controlled sequence of co-monomers, defined as letters of an alphabet, and are used to store information at the molecular level. Reading such messages is hence a sequencing task that can be efficiently achieved by tandem mass spectrometry. To improve their readability, structure of sequence-controlled synthetic polymers can be optimized, based on considerations regarding their fragmentation behavior. This strategy is described here for poly(phosphodiester)s, which were synthesized as monodisperse chains with more than 100 units but exhibited extremely complex dissociation spectra. In these polymers, two repeating units that differ by a simple H/CH variation were defined as the 0 and 1 bit of the ASCII code and spaced by a phosphate moiety. They were readily ionized in negative ion mode electrospray but dissociated via cleavage at all phosphate bonds upon collisional activation. Although allowing a complete sequence coverage of digital poly(phosphodiester)s, this fragmentation behavior was not efficient for macromolecules with more than 50 co-monomers, and data interpretation was very tedious. The structure of these polymers was then modified by introducing alkoxyamine linkages at appropriate location throughout the chain. A first design consisted of placing these low dissociation energy bonds between each monomeric bit: while cleavage of this sole bond greatly simplified MS/MS spectra, efficient sequencing was limited to chains with up to about 50 units. In contrast, introduction of alkoxyamine bonds between each byte (i.e. a set of eight co-monomers) was a more successful strategy. Long messages (so far, up to 8 bytes) could be read in MS experiments, where single-byte containing fragments released during the first activation stage were further dissociated for sequencing. The whole sequence of such byte-truncated poly(phosphodiester)s could be easily re-constructed based on a mass tagging system which permits to determine the original location of each byte in the chain. Copyright © 2017 John Wiley & Sons, Ltd.

Desorption Electrospray Ionization (DESI) of Digital Polymers: Direct Tandem Mass Spectrometry Decoding and Imaging from Materials Surfaces

“…We purposely chose two oligomers (P7 and P8 in Table 1) with the same mass but different sequence to draw each digit so that different readouts can be achieved depending on the reading mode (MS vs MS/MS). The design of these sequence‐defined oligomers was optimized so that all repeating units can be simultaneously ionized via phosphate deprotonation, a key condition to achieve the simplest MS/MS pattern, [ 13,52 ] with exclusive homolysis of alkoxyamine bonds between repeating units leading to a useful ranking of c fragments (Figure S5, Supporting Information). DESI exhibits similar propensity to multiple charging as compared to ESI, so the 3‐mers P7 and P8 (1563.9 Da) could readily be generated as triply deprotonated species ( m/ z 525.0) by DESI.…”

“…To address this issue, the chemical structure of monomers can be optimized to decrease their MS/MS reactivity and hence lower the number of dissociation pathways per repeating unit. [ 13,14 ] Using this strategy, the longest chain read so far by MS/MS alone is a 77‐mer. [ 4 ] Increasing the number of encoded bits per monomer is another efficient strategy to enhance the storage capacity of a single chain.…”

High definition polyphosphoesters: between nucleic acids and plastics

“…Different approaches have been taken to improve this situation. 137 Poly(phosphate-alkoxyamine) systems (Fig. 7c) made through alternating phosphoramidite and radical-radical couplings, enabled this through the spontaneous homolytic cleavage at the alkoxyamine site under mild conditions, resulting in an easily interpreted using secondary ion mass spectrometry (MS 2 ).…”

Cleavable Binary Dyads: Simplifying Data Extraction and Increasing Storage Density in Digital Polymers

“…The polymers have therefore an increased storage density in comparison to earlier design but do not lead to more complex MS/MS spectra since weak alkoxyamine bonds (i.e. only one per repeat unit) break preferentially during sequencing . Thus, this simple molecular design allows dyad detection and consequently greatly simplifies MS/MS data extraction from digital macromolecules.…”