Congratulations, Philip!

Congrats to Ashley Ogorek and @shubha-pani.bsky.social‬, who co-led this study, as well as the entire team: Eli, Jelena, Yichong, Fernando, Rachel, @xuhuihuangchem.bsky.social‬. This was a really fun collaboration with ‪@chembiobryan.bsky.social‬ - stay tuned for more to come!

/end

The enhanced bioorthogonality of the bCP ester enabled us to perform spatially-resolved RNA proximity labeling and RNA sequencing, including in human cells lines with high endogenous esterase activity.

9/n

The pCP / evolved BS2 pair performs well in multiple sub-compartments of mammalian cells.

8/n

We teamed up with @xuhuihuangchem.bsky.social to perform structural modeling and substrate docking to gain insights into the beneficial mutations.

7/n

Using DEEPMACh, we evolved BS2 esterase to increase activity toward pCP esters more than 230-fold.

6/n

To overcome this challenge, we developed a new platform, Directed Evolution of Enzymes via Masked Acid Chloride Probes ("DEEPMACh”). DEEPMACh combines yeast surface display with masked acylating probes, enabling rapid screening of >40 million enzyme mutants.

5/n

…BS2 esterase shows very low activity toward the pCP ester.

4/n

The methylcyclopropyl (mCP) ester protecting group together with BS2 esterase has been applied as a bioorthogonal system, but background unmasking of mCP occurs in mammalian cells. We found that the bulkier phenylCP group was much more bioorthogonal! However…

3/n

Context: combining bioorthogonal protecting groups with localized catalysts that unmask them is a powerful approach to modulate molecular activity. However, existing protecting groups are insufficiently bioorthogonal, or the catalysts that unmask them cannot be genetically targeted. 2/n

Thanks, Neel!

Thanks, Chang!

Thank you! We didn't run into mixing/order of addition issues. Nearly all components are already on DNA, and we add Cu last to commence the reaction. For mixing, we did vortexing then centrifugation, which worked well for our 20 uL scale reactions.

Congratulations, Phill! Very well-deserved!

Thanks especially to Matt and Beck for collaborating with us to merge our platform with data science and ML, and to Josh and Daniel (‪@coonlab.bsky.social‬) for performing essential mass spec characterization of the DNA-small molecule conjugates.‬‬

/end

Big congrats to co-1st authors Caleb Cox and @edwardpimentel.bsky.social‬ who showed incredible persistence and creativity, and to all co-authors: Beck Miller, Daniel Nesbitt, Justice LeMonds, @ethan-hartman-125.bsky.social‬, Tate Hancock, Robert Kennedy, Josh Coon, and Matt Sigman.

11/n

Overall, we believe this platform opens fundamentally new opportunities in data-driven discovery, optimization, and mechanistic understanding of synergistic catalytic reactions. There are many new directions we’re excited to explore – stay tuned for more in the coming years!

10/n

Excitingly, we observed correlation between ML predicted DNA nanoscaffold yields and experimental DNA-free reactions, including for kinetic time courses and for reactants not represented in the original DNA nanoscaffold library.

9/n

We teamed up with Matt Sigman and Beck Miller to use data science in library design and to combine our datasets with ML to generate predictive models. The model covers 18,000 reactant combinations under many reaction conditions.

8/n