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1. Next generation synthetic memory via intercepting recombinase function

   Short, A.E. (October 2023, Nature communications)

   Ross Cloney (Ed.)

   Here we present a technology to facilitate synthetic memory in a living system via repurposing Transcriptional Programming (i.e., our decision-making technology) parts, to regulate (intercept) recombinase function post-translation. We show that interception synthetic memory can facilitate programmable loss-of-function via site-specific deletion, programmable gain-of-function by way of site-specific inversion, and synthetic memory operations with nested Boolean logical operations. We can expand interception synthetic memory capacity more than 5-fold for a single recombinase, with reconfiguration specificity for multiple sites in parallel. Interception synthetic memory is ~10-times faster than previous generations of recombinase-based memory. We posit that the faster recombination speed of our next-generation memory technology is due to the post-translational regulation of recombinase function. This iteration of synthetic memory is complementary to decision-making via Transcriptional Programming – thus can be used to develop intelligent synthetic biological systems for myriad applications. 

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2. Next generation synthetic memory via intercepting recombinase function

   https://doi.org/10.1038/s41467-023-41043-w  

   Short, Andrew E.; Kim, Dowan; Milner, Prasaad T.; Wilson, Corey J. (December 2023, Nature Communications)

   Abstract Here we present a technology to facilitate synthetic memory in a living system via repurposing Transcriptional Programming (i.e., our decision-making technology) parts, to regulate (intercept) recombinase function post-translation. We show that interception synthetic memory can facilitate programmable loss-of-function via site-specific deletion, programmable gain-of-function by way of site-specific inversion, and synthetic memory operations with nested Boolean logical operations. We can expand interception synthetic memory capacity more than 5-fold for a single recombinase, with reconfiguration specificity for multiple sites in parallel. Interception synthetic memory is ~10-times faster than previous generations of recombinase-based memory. We posit that the faster recombination speed of our next-generation memory technology is due to the post-translational regulation of recombinase function. This iteration of synthetic memory is complementary to decision-making via Transcriptional Programming – thus can be used to develop intelligent synthetic biological systems for myriad applications. 

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3. Long-duration environmental biosensing by recording analyte detection in DNA using recombinase memory

   https://doi.org/10.1128/aem.02363-23  

   Kalvapalle, Prashant Bharadwaj; Sridhar, Swetha; Silberg, Jonathan J; Stadler, Lauren B (April 2024, Applied and Environmental Microbiology)

   Rudi, Knut (Ed.)

   ABSTRACT Microbial biosensors that convert environmental information into real-time visual outputs are limited in their sensing abilities in complex environments, such as soil and wastewater, due to optical inaccessibility. Biosensors that could record transient exposure to analytes within a large time window for later retrieval represent a promising approach to solve the accessibility problem. Here, we test the performance of recombinase-memory biosensors that sense a sugar (arabinose) and a microbial communication molecule (3-oxo-C12-L-homoserine lactone) over 8 days (~70 generations) following analyte exposure. These biosensors sense the analyte and trigger the expression of a recombinase enzyme which flips a segment of DNA, creating a genetic memory, and initiates fluorescent protein expression. The initial designs failed over time due to unintended DNA flipping in the absence of the analyte and loss of the flipped state after exposure to the analyte. Biosensor performance was improved by decreasing recombinase expression, removing the fluorescent protein output, and using quantitative PCR to read out stored information. Application of memory biosensors in wastewater isolates achieved memory of analyte exposure in an uncharacterizedPseudomonasisolate. By returning these engineered isolates to their native environments, recombinase-memory systems are expected to enable longer duration andin situinvestigation of microbial signaling, cross-feeding, community shifts, and gene transfer beyond the reach of traditional environmental biosensors.IMPORTANCEMicrobes mediate ecological processes over timescales that can far exceed the half-lives of transient metabolites and signals that drive their collective behaviors. We investigated strategies for engineering microbes to stably record their transient exposure to a chemical over many generations through DNA rearrangements. We identify genetic architectures that improve memory biosensor performance and characterize these in wastewater isolates. Memory biosensors are expected to be useful for monitoring cell-cell signals in biofilms, detecting transient exposure to chemical pollutants, and observing microbial cross-feeding through short-lived metabolites within cryptic methane, nitrogen, and sulfur cycling processes. They will also enablein situstudies of microbial responses to ephemeral environmental changes, or other ecological processes that are currently challenging to monitor non-destructively using real-time biosensors and analytical instruments. 

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4. Heterochiral modifications enhance robustness and function of DNA in living human cells

   https://doi.org/10.1002/cbic.202300755  

   Mallette, Tracy L.; Lidke, Diane S.; Lakin, Matthew R. (March 2024, ChemBioChem)

   Abstract Oligonucleotide therapeutics are becoming increasingly important as more are approved by the FDA, both for treatment and vaccination. Similarly, dynamic DNA nanotechnology is a promising technique that can be used to sense exogenous input molecules or endogenous biomarkers and integrate the results of multiple sensing reactionsin situvia a programmed cascade of reactions. The combination of these two technologies could be highly impactful in biomedicine by enabling smart oligonucleotide therapeutics that can autonomously sense and respond to a disease state. A particular challenge, however, is the limited lifetime of standard nucleic acid components in living cells and organisms due to degradation by endogenous nucleases. In this work, we address this challenge by incorporating mirror‐image, ʟ‐DNA nucleotides to produce heterochiral “gapmers”. We use dynamic DNA nanotechnology to show that these modifications keep the oligonucleotide intact in living human cells for longer than an unmodified strand. To this end, we used a sequential transfection protocol for delivering multiple nucleic acids into living human cells while providing enhanced confidence that subsequent interactions are actually occurring within the cells. Taken together, this work advances the state of the art of ʟ‐nucleic acid protection of oligonucleotides and DNA circuitry for applicationsin vivo. 

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5. RAD51 regulates eukaryotic chromatin motions in the absence of DNA damage

   https://doi.org/10.1091/mbc.E24-04-0188  

   Maarouf, Amine; Iqbal, Fadil; Sanaullah, Sarvath; Locatelli, Maëlle; Atanasiu, Andrew T; Kolbin, Daniel; Hommais, Chloé; Mühlemann, Joëlle K; Bonin, Keith; Bloom, Kerry; et al (November 2024, Molecular Biology of the Cell)

   Misteli, Tom (Ed.)

   In yeasts and higher eukaryotes, chromatin motions may be tuned to genomic functions, with transcriptional activation and the DNA damage response both leading to profound changes in chromatin dynamics. The RAD51 recombinase is a key mediator of chromatin mobility following DNA damage. As functions of RAD51 beyond DNA repair are being discovered, we asked whether RAD51 modulates chromatin dynamics in the absence of DNA damage and found that inhibition or depletion of RAD51 alters chromatin motions in undamaged cells. Inhibition of RAD51 increased nucleosome clustering. Predictions from polymer models are that chromatin clusters reduce chain mobility and, indeed, we measured reduced motion of individual chromatin loci in cells treated with a RAD51 inhibitor. This effect was conserved in mammalian cells, yeasts, and plant cells. In contrast, RAD51 depletion or inhibition increased global chromatin motions at the microscale. The results uncover a role for RAD51 in regulating local and global chromatin dynamics independently from DNA damage and highlight the importance of considering different physical scales when studying chromatin dynamics. 

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