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Detection of cytoplasmic DNA represents one of the most fundamental mechanisms of the innate immune system to sense the presence of microbial pathogens (1). Moreover, erroneous detection of endogenous DNA by the same sensing mechanisms has an important pathophysiological role in certain sterile inflammatory conditions (2,3). The endoplasmic-reticulum-resident protein STING is critically required for the initiation of type I interferon signalling upon detection of cytosolic DNA of both exogenous and endogenous origin (4-8). Next to its pivotal role in DNA sensing, STING also serves as a direct receptor for the detection of cyclic dinucleotides, which function as second messenger molecules in bacteria (9-13). DNA recognition, however, is triggered in an indirect fashion that depends on a recently characterized cytoplasmic nucleotidyl transferase, termed cGAMP synthase (cGAS), which upon interaction with DNA synthesizes a dinucleotide molecule that in turn binds to and activates STING (14,15). We here show in vivo and in vitro that the cGAS-catalysed reaction product is distinct from previously characterized cyclic dinucleotides. Using a combinatorial approach based on mass spectrometry, enzymatic digestion, NMR analysis and chemical synthesis we demonstrate that cGAS produces a cyclic GMP-AMP dinucleotide, which comprises a 2'-5' and a 3'-5' phosphodiester linkage >Gp(2'-5')Ap(3'-5')>. We found that the presence of this 2'-5' linkage was required to exert potent activation of human STING. Moreover, we show that cGAS first catalyses the synthesis of a linear 2' -5'-linked dinucleotide, which is then subject to cGAS-dependent cyclization in a second step through a 3'-5' phosphodiester linkage. This 13-membered ring structure defines a novel class of second messenger molecules, extending the family of 2' -5'-linked antiviral biomolecules., Recently, it has been demonstrated that upon intracellular DNA delivery, a cytoplasmic enzyme dubbed cyclic GMP-AMP synthase (cGAS) produces a ribo-dinucleotide, which in turn binds to and activates STING (14,15). [...]

A study explores the use for the synthesis of alpha-P-modified triphosphate (NTP) analogues in conjunction with ethylenediamine, which act as a dephosphorylating reagent on basis of Ludwig-Eckstein approach the successful preparation of various alpha-P-modified NTP analogues. An efficient and convenient protocol to synthesis alpha-P-modified nucleoside di- analogues that are otherwise difficult to obtain is developed.

Preparation of synthetic RNA-like molecules with catalytic activity resembling that of the hammer head ribozyme has been investigated as a potential pathway for the generation of truncated catalysts. The loss of cleavage activity is negligible when the neutral hexaethylene glycol linker is used to replace all the nucleotides of loop II and two of the base pairs of stem II. As three or all four of the base paris of stem II are eliminated, certain loss of activity occurs, but these molecules continue to be active RNA cleaving molecules.

RIG-I is a cytosolic helicase that senses 5′-ppp RNA contained in negative-strand RNA viruses and triggers innate antiviral immune responses. Calorimetric binding studies established that the RIG-I C-terminal regulatory domain (CTD) binds to blunt-end double-stranded 5′-ppp RNA a factor of 17 more tightly than to its single-stranded counterpart. Here we report on the crystal structure of RIG-I CTD bound to both blunt ends of a self-complementary 5′-ppp dsRNA 12-mer, with interactions involving 5′-pp clearly visible in the complex. The structure, supported by mutation studies, defines how a lysine-rich basic cleft within the RIG-I CTD sequesters the observable 5′-pp of the bound RNA, with a stacked phenylalanine capping the terminal base pair. Key intermolecular interactions observed in the crystalline state are retained in the complex of 5′-ppp dsRNA 24-mer and full-length RIG-I under in vivo conditions, as evaluated from the impact of binding pocket RIG-I mutations and 2′-OCH3 RNA modifications on the interferon response. [ABSTRACT FROM AUTHOR]