Thermodynamic bounds on ultrasensitivity in covalent switching.

Switch-like motifs are among the basic building blocks of biochemical networks. A common motif that can serve as an ultrasensitive switch consists of two enzymes acting antagonistically on a substrate, one making and the other removing a covalent modification. To work as a switch, such covalent modification cycles must be held out of thermodynamic equilibrium by continuous expenditure of energy. Here, we exploit the linear framework for timescale separation to establish tight bounds on the performance of any covalent-modification switch in terms of the chemical potential difference driving the cycle. The bounds apply to arbitrary enzyme mechanisms, not just Michaelis-Menten, with arbitrary rate constants and thereby reflect fundamental physical constraints on covalent switching.

Scalable co-sequencing of RNA and DNA from individual nuclei.

The ideal technology for directly investigating the relationship between genotype and phenotype would analyze both RNA and DNA genome-wide and with single-cell resolution. However, existing tools lack the throughput required for comprehensive analysis of complex tumors and tissues. We introduce a highly scalable method for jointly profiling DNA and expression following nucleosome depletion (DEFND-seq). In DEFND-seq, nuclei are nucleosome-depleted, tagmented, and separated into individual droplets for mRNA and genomic DNA barcoding. Once nuclei have been depleted of nucleosomes, subsequent steps can be performed using the widely available 10x Genomics droplet microfluidic technology and commercial kits without experimental modification. We demonstrate the production of high-complexity mRNA and gDNA sequencing libraries from thousands of individual nuclei from both cell lines and archived surgical specimens for associating gene expression phenotypes with both copy number and single nucleotide variants.