Engineered reversible inhibition of SpyCatcher reactivity enables rapid generation of bispecific antibodies.

The precise regulation of protein function is essential in biological systems and a key goal in chemical biology and protein engineering. Here, we describe a straightforward method to engineer functional control into the isopeptide bond-forming SpyTag/SpyCatcher protein ligation system. First, we perform a cysteine scan of the structured region of SpyCatcher.

Trivalent rare earth metal cofactors confer rapid NP-DNA polymerase activity.

A DNA polymerase with a single mutation and a divalent calcium cofactor catalyzes the synthesis of unnatural N3'→P5' phosphoramidate (NP) bonds to form NP-DNA. However, this template-directed phosphoryl transfer activity remains orders of magnitude slower than native phosphodiester synthesis. Here, we used time-resolved x-ray crystallography to show that NP-DNA synthesis proceeds with a single detectable calcium ion in the active site.

A Potential Role for Aminoacylation in Primordial RNA Copying Chemistry.

Aminoacylated tRNAs are the substrates for ribosomal protein synthesis in all branches of life, implying an ancient origin for aminoacylation chemistry. In the 1970s, Orgel and colleagues reported potentially prebiotic routes to aminoacylated nucleotides and their RNA-templated condensation to form amino acid-bridged dinucleotides. However, it is unclear whether such reactions would have aided or impeded non-enzymatic RNA replication.

Structural interpretation of the effects of threo-nucleotides on nonenzymatic template-directed polymerization.

The prebiotic synthesis of ribonucleotides is likely to have been accompanied by the synthesis of noncanonical nucleotides including the threo-nucleotide building blocks of TNA. Here, we examine the ability of activated threo-nucleotides to participate in nonenzymatic template-directed polymerization. We find that primer extension by multiple sequential threo-nucleotide monomers is strongly disfavored relative to ribo-nucleotides.

Synthesis of phosphoramidate-linked DNA by a modified DNA polymerase.

All known polymerases copy genetic material by catalyzing phosphodiester bond formation. This highly conserved activity proceeds by a common mechanism, such that incorporated nucleoside analogs terminate chain elongation if the resulting primer strand lacks a terminal hydroxyl group. Even conservatively substituted 3'-amino nucleotides generally act as chain terminators, and no enzymatic pathway for their polymerization has yet been found.

DNA polymerase activity on synthetic N3'→P5' phosphoramidate DNA templates.

Genetic polymers that could plausibly govern life in the universe might inhabit a broad swath of chemical space. A subset of these genetic systems can exchange information with RNA and DNA and could therefore form the basis for model protocells in the laboratory. N3'→P5' phosphoramidate (NP) DNA is defined by a conservative linkage substitution and has shown promise as a protocellular genetic material, but much remains unknown about its functionality and fidelity due to limited enzymatic tools.

Inosine, but none of the 8-oxo-purines, is a plausible component of a primordial version of RNA.

The emergence of primordial RNA-based life would have required the abiotic synthesis of nucleotides, and their participation in nonenzymatic RNA replication. Although considerable progress has been made toward potentially prebiotic syntheses of the pyrimidine nucleotides (C and U) and their 2-thio variants, efficient routes to the canonical purine nucleotides (A and G) remain elusive. Reported syntheses are low yielding and generate a large number of undesired side products.

Comparative analysis of LIN28-RNA binding sites identified at single nucleotide resolution.

It remains a formidable challenge to characterize the diverse complexes of RNA binding proteins and their targets. While crosslink and immunoprecipitation (CLIP) methods are powerful techniques that identify RNA targets on a global scale, the resolution and consistency of these methods is a matter of debate. Here we present a comparative analysis of LIN28-pre-let-7 UV-induced crosslinking using a tandem mass spectrometry (MS/MS) and deep sequencing interrogation of in vitro crosslinked complexes.

Enhanced Nonenzymatic RNA Copying with 2-Aminoimidazole Activated Nucleotides.

Achieving efficient nonenzymatic replication of RNA is an important step toward the synthesis of self-replicating protocells that may mimic early forms of life. Despite recent progress, the nonenzymatic copying of templates containing mixed sequences remains slow and inefficient. Here we demonstrate that activating nucleotides with 2-aminoimidazole results in superior reaction kinetics and improved yields of primer extension reaction products.

N-Carboxyanhydride-Mediated Fatty Acylation of Amino Acids and Peptides for Functionalization of Protocell Membranes.

Early protocells are likely to have arisen from the self-assembly of RNA, peptide, and lipid molecules that were generated and concentrated within geologically favorable environments on the early Earth. The reactivity of these components in a prebiotic environment that supplied sources of chemical energy could have produced additional species with properties favorable to the emergence of protocells. The geochemically plausible activation of amino acids by carbonyl sulfide has been shown to generate short peptides via the formation of cyclic amino acid N-carboxyanhydrides (NCAs).

Molecular fMRI of Serotonin Transport.

Reuptake of neurotransmitters from the brain interstitium shapes chemical signaling processes and is disrupted in several pathologies. Serotonin reuptake in particular is important for mood regulation and is inhibited by first-line drugs for treatment of depression. Here we introduce a molecular-level fMRI technique for micron-scale mapping of serotonin transport in live animals.

Experimental and Computational Evidence for a Loose Transition State in Phosphoroimidazolide Hydrolysis.

Phosphoroimidazolides play a critical role in several enzymatic phosphoryl transfer reactions and have been studied extensively as activated monomers for nonenzymatic nucleic acid replication, but the detailed mechanisms of these phosphoryl transfer reactions remain elusive. Some aspects of the mechanism can be deduced by studying the hydrolysis reaction, a simpler system that is amenable to a thorough mechanistic treatment. Here we characterize the transition state of phosphoroimidazolide hydrolysis by kinetic isotope effect (KIE) and linear free energy relationship (LFER) measurements, and theoretical calculations.

Pinpointing RNA-Protein Cross-Links with Site-Specific Stable Isotope-Labeled Oligonucleotides.

High affinity RNA-protein interactions are critical to cellular function, but directly identifying the determinants of binding within these complexes is often difficult. Here, we introduce a stable isotope mass labeling technique to assign specific interacting nucleotides in an oligonucleotide-protein complex by photo-cross-linking. The method relies on generating site-specific oxygen-18-labeled phosphodiester linkages in oligonucleotides, such that covalent peptide-oligonucleotide cross-link sites arising from ultraviolet irradiation can be assigned to specific sequence positions in both RNA and protein simultaneously by mass spectrometry.

Bidirectional Direct Sequencing of Noncanonical RNA by Two-Dimensional Analysis of Mass Chromatograms.

Mass spectrometry (MS) is a powerful technique for characterizing noncanonical nucleobases and other chemical modifications in small RNAs, yielding rich chemical information that is complementary to high-throughput indirect sequencing. However, mass spectra are often prohibitively complex when fragment ions are analyzed following either solution phase hydrolysis or gas phase fragmentation. For all but the simplest cases, ions arising from multiple fragmentation events, alternative fragmentation pathways, and diverse salt adducts frequently obscure desired single-cut fragment ions.

Molecular-level functional magnetic resonance imaging of dopaminergic signaling.

We demonstrate a technique for mapping brain activity that combines molecular specificity and spatial coverage using a neurotransmitter sensor detectable by magnetic resonance imaging (MRI). This molecular functional MRI (fMRI) method yielded time-resolved volumetric measurements of dopamine release evoked by reward-related lateral hypothalamic brain stimulation of rats injected with the neurotransmitter sensor. Peak dopamine concentrations and release rates were observed in the anterior nucleus accumbens core.

Magnetic nanosensors optimized for rapid and reversible self-assembly.

Magnetic nanoparticle-based sensors for MRI have been accelerated to a timescale of seconds using densely-functionalized particles of small size. Parameters that increase response rates also result in large nuclear magnetic relaxation rate and light scattering changes, allowing signals to be detected almost immediately after changes in calcium concentration.

Structure-guided directed evolution of highly selective p450-based magnetic resonance imaging sensors for dopamine and serotonin.

New tools that allow dynamic visualization of molecular neural events are important for studying the basis of brain activity and disease. Sensors that permit ligand-sensitive magnetic resonance imaging (MRI) are useful reagents due to the noninvasive nature and good temporal and spatial resolution of MR methods. Paramagnetic metalloproteins can be effective MRI sensors due to the selectivity imparted by the protein active site and the ability to tune protein properties using techniques such as directed evolution.

Metal-substituted protein MRI contrast agents engineered for enhanced relaxivity and ligand sensitivity.

Engineered metalloproteins constitute a flexible new class of analyte-sensitive molecular imaging agents detectable by magnetic resonance imaging (MRI), but their contrast effects are generally weaker than synthetic agents. To augment the proton relaxivity of agents derived from the heme domain of cytochrome P450 BM3 (BM3h), we formed manganese(III)-containing proteins that have higher electron spin than their native ferric iron counterparts. Metal substitution was achieved by coexpressing BM3h variants with the bacterial heme transporter ChuA in Escherichia coli and supplementing the growth medium with Mn3+-protoporphyrin IX.

Challenges for Molecular Neuroimaging with MRI.

Magnetic resonance (MRI)-based molecular imaging methods are beginning to have impact in neuroscience. A growing number of molecular imaging agents have been synthesized and tested in vitro, but so far relatively few have been validated in the brains of live animals. Here, we discuss key challenges associated with expanding the repertoire of successful molecular neuroimaging approaches.

A single protease, Apg4B, is specific for the autophagy-related ubiquitin-like proteins GATE-16, MAP1-LC3, GABARAP, and Apg8L.

Apg8 is a ubiquitin-like protein involved in autophagy in yeast. Apg8 is covalently but transiently attached to membrane lipids through the actions of activating, conjugating, and processing/deconjugating enzymes. The mammalian Apg8 homologues GATE-16, GARARAP, and MAP1-LC3 have been implicated in intra-Golgi transport, receptor sorting, and autophagy, respectively.