Click chemistry for rapid labeling and ligation of RNA

The copper(I)-promoted azide-alkyne cycloaddition reaction (click chemistry) is shown to be compatible with RNA (with free 2'-hydroxyl groups) in spite of the intrinsic lability of RNA. RNA degradation is minimized through stabilization of the Cu(I) in aqueous buffer with acetonitrile as cosolvent and no other ligand; this suggests the general possibility of "ligandless" click chemistry. With the viability of click chemistry validated on synthetic RNA bearing "click"-reactive alkynes, the scope of the reaction is extended to in-vitro-transcribed or, indeed, any RNA, as a click-reactive azide is incorporated enzymatically. Once clickable groups are installed on RNA, they can be rapidly click labeled or conjugated together in click ligations, which may be either templated or nontemplated. In click ligations the resultant unnatural triazole-linked RNA backbone is not detrimental to RNA function, thus suggesting a broad applicability of click chemistry in RNA biological studies.     

Regio‐Selective Chemical‐Enzymatic Synthesis of Pyrimidine Nucleotides Facilitates RNA Structure and Dynamics Studies

Isotope labeling has revolutionized NMR studies of small nucleic acids, but to extend this technology to larger RNAs, site‐specific labeling tools to expedite NMR structural and dynamics studies are required. Using enzymes from the pentose phosphate pathway, we coupled chemically synthesized uracil nucleobase with specifically ¹³C‐labeled ribose to synthesize both UTP and CTP in nearly quantitative yields. This chemoenzymatic method affords a cost‐effective preparation of labels that are unattainable by current methods. The methodology generates versatile ¹³C and ¹⁵N labeling patterns which, when employed with relaxation‐optimized NMR spectroscopy, effectively mitigate problems of rapid relaxation that result in low resolution and sensitivity. The methodology is demonstrated with RNAs of various sizes, complexity, and function: the exon splicing silencer 3 (27 nt), iron responsive element (29 nt), Pro‐tRNA (76 nt), and HIV‐1 core encapsidation signal (155 nt).     

Chemical reporters for monitoring RNA synthesis and poly(A) tail dynamics

A versatile "clickable" nucleoside: Metabolic labeling of cells is useful in studying the dynamics of biological molecules. N(6) pA can be utilized by all three mammalian RNA polymerases, as well as poly(A) polymerase. Because of its alkyne modification, RNA labeled with N(6) pA can be visualized and purified by using click chemistry.     

Cleavage of an RNA Model Compound by an Arylmercury Complex

A water-soluble arylmercury complex has been synthesized, and its ability to catalyze the cleavage of the phosphodiester linkage of the RNA model compound adenylyl-3′,5′-(2′,3′-O-methyleneadenosine) has been assessed over a pH range of 3–8.5 and a catalyst concentration range of 0–7 mM. In the presence of 1 mM catalyst, the observed pH–rate profile featured a new pH-independent region between pH 6 and 7, the catalyzed reaction being as much as eight times faster than the background reaction. At pH 7, the acceleration increased linearly from three- to 17-fold upon increasing the catalyst concentration from 1 to 7 mM. The linear dependence indicates a relatively low affinity of the catalyst for the substrate and, hence, the potential for considerable improvement on tethering to an appropriate targeting group, such as an oligonucleotide.     

Aptamer to ribozyme: the intrinsic catalytic potential of a small RNA

The discovery of RNA-based catalysis 23 years ago dramatically changed the way biologists and biochemists thought of RNA. In the recent past, several ribozymes structures have provided some answers as to how catalysis is accomplished and how it relates to RNA structure and folding. However, there is still little information as to how catalytic activity evolved. Here we show that the small malachite green-binding aptamer has intrinsic catalytic potential that can be realized by designing the proper substrate. The charge distribution within the RNA binding pocket stabilizes the transition state of an ester hydrolysis reaction and thus accelerates the overall reaction. The results suggest that electrostatic forces can contribute significantly to RNA-based catalysis. Moreover, even simple RNA structures that have not been selected for catalytic properties can have a basic catalytic potential if they encounter the right substrate. This provides a possible starting point for the molecular evolution of more complex ribozymes.     

Practical Synthesis of Cap-4 RNA

Eukaryotic mRNAs possess 5′ caps that are determinants for their function. A structural characteristic of 5′ caps is methylation, with this feature already present in early eukaryotes such as Trypanosoma. While the common cap-0 (m⁷GpppN) shows a rather simple methylation pattern, the Trypanosoma cap-4 displays seven distinguished additional methylations within the first four nucleotides. The study of essential biological functions mediated by these unique structural features of the cap-4 and thereby of the metabolism of an important class of human pathogenic parasites is hindered by the lack of reliable preparation methods. Herein we describe the synthesis of custom-made nucleoside phosphoramidite building blocks for m⁶₂Am and m³Um, their incorporation into short RNAs, the efficient construction of the 5′-to-5′ triphosphate bridge to guanosine by using a solid-phase approach, the selective enzymatic methylation at position N7 of the inverted guanosine, and enzymatic ligation to generate trypanosomatid mRNAs of up to 40 nucleotides in length. This study introduces a reliable synthetic strategy to the much-needed cap-4 RNA probes for integrated structural biology studies, using a combination of chemical and enzymatic steps.     

Characterization of the Rv3377c Gene Product,a Type‐B Diterpene Cyclase,from the Mycobacterium tuberculosis H37 Genome

The Rv3377c gene from the Mycobacterium tuberculosis H37 genome is specifically limited to those Mycobacterium species that cause tuberculosis. We have demonstrated that the gene product of Rv3377c is a diterpene cyclase that catalyzes the formation of tuberculosinol from geranylgeranyl diphosphate (GGPP). However, the characteristics of this enzyme had not previously been studied in detail with homogeneously purified enzyme. The purified enzyme catalyzed the synthesis of tuberculosinyl diphosphate from GGPP, but it did not bring about the synthesis of tuberculosinol. Optimal conditions for the highest activity were found to be as follows: pH 7.5, 30 °C, Mg^II (0.1 mM ), and Triton X‐100 (0.1 %). Under these conditions, the kinetic values of K_M and k_cat were determined to be 11.7±1.9 μM for GGPP and 12.7±0.7 min^?1, respectively, whereas the specific activity was 186 nmol min^?1 mg^?1. The enzyme activity was inhibited at substrate concentrations higher than 50 μM . The catalytic activity was strongly inhibited by 15‐aza‐dihydrogeranylgeraniol and 5‐isopropyl‐N,N,N,2‐tetramethyl‐4‐(piperidine‐1‐carbonyloxy)benzenaminium chloride (Amo‐1618). The DXDTT_293–297 motif, corresponding to the DXDDTA motif conserved among terpene cyclases, was mutated in order to investigate its function. The middle D295 was found to be the most crucial entity for the catalysis. D293 and two threonine residues function synergistically to enhance the acidity of D295, possibly through hydrogen‐bonding networks. The Rv3377c enzyme could also react with (14R/S)‐14,15‐oxidoGGPP to generate 3α‐ and 3β‐hydroxytuberculosinyl diphosphate. Conformational analyses were carried out with deuterium‐labeled GGPP and oxidoGGPP. We found that GGPP and (14R)‐oxidoGGPP adopted a chair/chair conformation, but (14S)‐oxidoGGPP adopted a boat/chair conformation. Interestingly, the conformations of oxidoGGPP for the A‐ring formation are the opposite of those of oxidosqualene when it is used as a substrate by squalene cyclases for the biosynthesis of hopene and tetrahymanol. (3R)‐Oxidosqualene is folded in a boat conformation, whereas (3S)‐2,3‐oxidosqualene folds into a chair conformation, for the formation of the A‐rings of the hopene and tetrahymanol skeletons, respectively.     

Biotinylated Phosphoproteins from Kinase‐Catalyzed Biotinylation are Stable to Phosphatases: Implications for Phosphoproteomics

Kinase‐catalyzed protein phosphorylation is involved in a wide variety of cellular events. Development of methods to monitor phosphorylation is critical to understand cell biology. Our lab recently discovered kinase‐catalyzed biotinylation, where ATP‐biotin is utilized by kinases to label phosphopeptides or phosphoproteins with a biotin tag. To exploit kinase‐catalyzed biotinylation for phosphoprotein purification and identification in a cellular context, the susceptibility of the biotin tag to phosphatases was characterized. We found that the phosphorylbiotin group on peptide and protein substrates was relatively insensitive to protein phosphatases. To understand how phosphatase stability would impact phosphoproteomics research applications, kinase‐catalyzed biotinylation of cell lysates was performed in the presence of kinase or phosphatase inhibitors. We found that biotinylation with ATP‐biotin was sensitive to inhibitors, although with variable effects compared to ATP phosphorylation. The results suggest that kinase‐catalyzed biotinylation is well suited for phosphoproteomics studies, with particular utility towards monitoring low‐abundance phosphoproteins or characterizing the influence of inhibitor drugs on protein phosphorylation.     

An Unexpected Promiscuous Activity of 4-Oxalocrotonate Tautomerase: The cis-trans Isomerisation of Nitrostyrene

Serendipitous switch: While exploring cis-nitrostyrene as a potential electrophile in Michael-type addition reactions catalysed by the enzyme 4-oxalocrotonate tautomerase (4-OT), it was unexpectedly found that 4-OT catalyses the isomerisation of cis-nitrostyrene to trans-nitrostyrene (k(cat) /K(m) = 1.9×10(3) M(-1) s(-1) ).