研究実績の概要 |
Linear sequences encoding the combox and ribox ribozymes have been used to perform in vitro transcription to generate an water-in-oil nanoemulsion containing in each droplet transcribed ribozyme as well as the reaction components required for the ribozyme-dependent reduction of benzyl alcohol and a diaphorase-resazurin-based coupled system for the detection of the subsequent NADH oxidation. Nanoemulsions were prepared with both the original sequences and libraries generated through mutagenic PCR where sequences of 20 or 30 random nucleotides were introduced near the described substrate-binding pocket. The nanoemulsions were analyzed with a microfluidics system, aiming to detect the fluorescence of resorufin generated if NADH is oxidized.
The analysis of nanodroplets revealed that the droplets containing only original sequences did not display resorufin fluorescence, consistent with the fact that they are not able to perform multiple catalytic cycles (or can only do so at very low rates). However, analysis of the droplets containing the sequences transcribed from libraries revealed a small population with high resorufin fluorescence intensity (not present in negative controls), potentially corresponding to variants with the ability to catalyze the target reaction in multiple turnover conditions. Nevertheless, attempts to amplify the variants contained in these droplets have not been successful, potentially due to the mode of binding of the variants to the beads enclosed in the droplets.
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今後の研究の推進方策 |
A first strategy will be to modify the binding mode of variants to beads to enable a more efficient amplification of variants after selection with the microfluidics system. To that extent, a longer linker region connecting the variants with the beads will be tested.
Alternatively, a common region with no variability will be added to the end of the sequences distal from the beads, and amplification from such invariable distal region will be tested.
As a different strategy, an in vivo selection system will also be tested. The in vivo selection system couples the generation of oxidized NAD+ with survivability of cells under anaerobic conditions, as described in Sells Vidal, Murray and Heap, 2021. Ribox/combox libraries will be cloned in a plasmid appropriate for in vivo transcription, and transformed into the selector strain. Transformed cells will be grown anaerobically in minimal media supplemented with benzaldehyde. After growth is observed, the sequences of the variants contained in the surviving cells will be analyzed to determine the optimal variants. If additional RNA stability is required, additional mutations will be introduced in the selector strains to knock-out RNases.
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