Cell Surface Labeling and Detection of Protein Tyrosine Kinase 7 via Covalent Aptamers.

Covalent aptamers are novel biochemical tools for fast and selective transfer of labels to target proteins. Equipped with cleavable electrophiles, these nucleic acid probes enable the installation of functional handles onto native proteins. The high affinity and specificity with which aptamers bind their selected targets allows for quick, covalent labeling that can compete with nuclease-mediated degradation. Here, we introduce the first application of covalent aptamers to modify a specific cell surface protein through proximity-driven label transfer. We targeted protein tyrosine kinase 7 (PTK7), a prominent cancer marker, and demonstrated aptamer-mediated biotin transfer to specific lysine residues on the extracellular domain of the protein. This allowed for tracking of PTK7 expression, localization, and cellular internalization. These studies validate the programmability of covalent aptamers and highlight their applicability in a cellular context, including protein and small molecule delivery.

Protein labeling and crosslinking by covalent aptamers.

In this chapter, a new approach to the selective modification of native proteins is discussed, using electrophilic covalent aptamers. These biochemical tools are generated through the site-specific incorporation of a label-transferring or crosslinking electrophile into a DNA aptamer. Covalent aptamers provide the ability to transfer a variety of functional handles to a protein of interest or to irreversibly crosslink to the target. Methods for the aptamer-mediated labeling and crosslinking of thrombin are described. Thrombin labeling is fast and selective, in both simple buffer and in human plasma and outcompetes nuclease-mediated degradation. This approach provides facile, sensitive detection of labeled protein by western blot, SDS-PAGE, and mass spectrometry.

Small-molecule control of neurotransmitter sulfonation.

Controlling unmodified serotonin levels in brain synapses is a primary objective when treating major depressive disorder-a disease that afflicts ∼20% of the world's population. Roughly 60% of patients respond poorly to first-line treatments and thus new therapeutic strategies are sought. To this end, we have constructed isoform-specific inhibitors of the human cytosolic sulfotransferase 1A3 (SULT1A3)-the isoform responsible for sulfonating ∼80% of the serotonin in the extracellular brain fluid. The inhibitor design includes a core ring structure, which anchors the inhibitor into a SULT1A3-specific binding pocket located outside the active site, and a side chain crafted to act as a latch to inhibit turnover by fastening down the SULT1A3 active-site cap. The inhibitors are allosteric, they bind with nanomolar affinity and are highly specific for the 1A3 isoform. The cap-stabilizing effects of the latch can be accurately calculated and are predicted to extend throughout the cap and into the surrounding protein. A free-energy correlation demonstrates that the percent inhibition at saturating inhibitor varies linearly with cap stabilization - the correlation is linear because the rate-limiting step of the catalytic cycle, nucleotide release, scales linearly with the fraction of enzyme in the cap-open form. Inhibitor efficacy in cultured cells was studied using a human mammary epithelial cell line that expresses SULT1A3 at levels comparable with those found in neurons. The inhibitors perform similarly in ex vivo and in vitro studies; consequently, SULT1A3 turnover can now be potently suppressed in an isoform-specific manner in human cells.

Allosteres to regulate neurotransmitter sulfonation.

Catecholamine neurotransmitter levels in the synapses of the brain shape human disposition-cognitive flexibility, aggression, depression, and reward seeking-and manipulating these levels is a major objective of the pharmaceutical industry. Certain neurotransmitters are extensively sulfonated and inactivated by human sulfotransferase 1A3 (SULT1A3). To our knowledge, sulfonation as a therapeutic means of regulating transmitter activity has not been explored. Here, we describe the discovery of a SULT1A3 allosteric site that can be used to inhibit the enzyme. The structure of the new site is determined using spin-label-triangulation NMR. The site forms a cleft at the edge of a conserved ∼30-residue active-site cap that must open and close during the catalytic cycle. Allosteres anchor into the site via π-stacking interactions with two residues that sandwich the planar core of the allostere and inhibit the enzyme through cap-stabilizing interactions with substituents attached to the core. Changes in cap free energy were calculated ab initio as a function of core substituents and used to design and synthesize a series of inhibitors intended to progressively stabilize the cap and slow turnover. The inhibitors bound tightly (34 nm to 7.4 µm) and exhibited progressive inhibition. The cap-stabilizing effects of the inhibitors were experimentally determined and agreed remarkably well with the theoretical predictions. These studies establish a reliable heuristic for the design of SULT1A3 allosteric inhibitors and demonstrate that the free-energy changes of a small, dynamic loop that is critical for SULT substrate selection and turnover can be calculated accurately.