Purine Recognition Research Articles

Sensing of viral lung infections by cGAS-STING

Lower respiratory tract infections caused over 4 million deaths per year worldwide, especially in low-income countries. Viral respiratory infections often occur as rapidly spreading seasonal endemic or epidemic, and sometimes due to new respiratory viruses including corona viruses. The first level of host defense against viral infection is based on the innate immune system and intracellular killing mechanisms. The latter is activated by the release of viral DNA or RNA into the cytosol of the infected cells during the initial phase of virus replication. Viral DNA and RNA are recognized by the cyclic guanosine monophosphate (cGMP)-adenosine monophosphate (AMP) synthase (cGAS)–stimulator of interferon (IFN) genes (STING) sensing pathway, leading to the activation of type-I and -III IFN synthesis, with the aim to limit viral replication. However, the efficacy of the cGAS-STING sensing mechanism seems to vary with different viruses, and therefore, so is the efficacy of the host defense mechanism. Viral DNA can be sensed by different proteins including DNA-dependent activator of IFN regulating factor (DAI), cGAS, and toll-like receptor-9 (TLR-9). Viral RNA is recognized by retinoid acid-inducible gene 1 (RIG-1), TLR-7 and TLR-8. The question if cGAS also recognizes viral RNA remains unclear. The activation of IFN synthesis by cGAS is initiated by the recognition of purines and pyrimidines and their enzymatic conversion into cGMP and cyclic AMP (cAMP), followed by the activation of STING. In addition, it is indicated that several viruses can evade the cGAS-STING signaling and escape the host defense. This review aims to summarize the role of cGAS-STING as a host defense mechanism against viral respiratory tract infections.

Layer-by-Layer Assembly of SAM-supported Porphyrin-based Metal Organic Frameworks for Molecular Recognition

The development of the sensing systems based on the ultrathin films of functional metal organic networks as efficient synthetic alternative to molecular biosensors is in the focus of intense research. Herein we report how to fabricate uniformly structured surface-attached metal organic frameworks (SURMOFs) from zinc complex of meso-carboxyphenyl-substituted porphyrin and zinc acetate as films on the surface of self-assembled monolayers of silanes and thiols. The layer-by-layer self-assembly with shortened time of the layer deposition at room temperature was applied to obtain 5-layer SURMOFs with high crystallinity, which was confirmed by X-ray diffraction. These SAM-supported SURMOF films were used as sensing layers for qualitative response to organic molecules in aqueous solutions. By using surface plasmon resonance spectroscopy, we showed that the SURMOFs do not bind 4-nitrophenol and uracil whereas they are capable of absorption of purine compounds such as adenine and adenosine 5-monophosphate. This binding is a size sensitive process in favor to adenine suggesting the effect of the SURMOF structure on the molecular recognition. The UV-vis spectroscopy study of the process indicated that the recognition of purines by the porphyrin-based SURMOF proceeds through the interactions of the amine groups with zinc ions in metal clusters rather than with the central metal ions in porphyrin cores. Our findings provide an insight into the sensing properties of the porphyrin-based SURMOFs that is useful for further advancements in fabrication of responsive thin films for biologically relevant analytes.

Tailoring Imprinted Titania Nanoparticles for Purines Recognition

Molecular imprinted titania nanoparticles were developed for selective recognition of purines, for example, guanine and its final oxidation product uric acid. Titania nanoparticles were prepared by hydrolysis of titanium butoxide as precursor in the presence of pattern molecules. The morphology of synthesized nanoparticles is evaluated by SEM images. Recognition characteristics of imprinted titania nanoparticles are studied by exposing them to standard solution of guanine and uric acid, respectively. The resultant change in their concentration is determined by UV/Vis analysis that indicated imprinted titania nanoparticles possess high affinity for print molecules. In both cases, nonimprinted titania is taken as control to observe nonspecific binding interactions. Cross sensitivity studies suggested that imprinted titania is at least five times more selective for binding print molecules than competing analyte thus indicating its potential for bioassay of purines.

Syntheses of Triostin A Antibiotic and Nucleobase‐Functionalized Analogs as New DNA Binders

AbstractA total synthesis of the natural product triostin A, wherein the N‐methylated depsipeptide scaffold is constructed by solution‐phase peptide chemistry followed by disulfide formation and macrocyclization, is described. Finally, the quinoxalines were attached to provide the DNA bisintercalator. Analogs of triostin A were obtained by the successive functionalization of the cyclic depsipeptide with pyrimidine or purine recognition units. The attachment of functional units was achieved by the orthogonal protection of the respective side chain amino functionalities. The nucleobase‐functionalized triostin analogs have the potential to recognize double‐stranded DNA by hydrogen bonding. The interaction with DNA was investigated by UV spectroscopy and fluorescence intercalator displacement. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)

Human UP1 as a model for understanding purine recognition in the family of proteins containing the RNA recognition motif (RRM).

Heterogeneous ribonucleoprotein A1 (hnRNP A1) is a prototype for the family of eukaryotic RNA processing proteins containing the common RNA recognition motif (RRM). The region consisting of residues 1-195 of hnRNP A1 is referred to as UP1. This region has two RRMs and has a high affinity for both single-stranded RNA and the human telomeric repeat sequence d(TTAGGG)(n). We have used UP1's novel DNA binding to investigate how RRMs bind nucleic acid bases through their highly conserved RNP consensus sequences. Nine complexes of UP1 bound to modified telomeric repeats were investigated using equilibrium fluorescence binding and X-ray crystallography. In two of the complexes, alteration of a guanine to either 2-aminopurine or nebularine resulted in an increase in K(d) from 88nM to 209nM and 316nM, respectively. The loss of these orienting interactions between UP1 and the substituted base allows it to flip between syn and anti conformations. Substitution of the same base with 7-deaza-guanine preserves the O6/N1 contacts but still increases the K(d) to 296nM and suggests that it is not simply the loss of affinity that gives rise to the base mobility, but also the stereochemistry of the specific contact to O6. Although these studies provide details of UP1 interactions to nucleic acids, three general observations about RRMs are also evident: (1) as suggested by informatic studies, main-chain to base hydrogen bonding makes up an important aspect of ligand recognition (2) steric clashes generated by modification of a hydrogen bond donor-acceptor pair to a donor-donor pair are poorly tolerated and (3) a conserved lysine position proximal to RNP-2 (K(106)-IFVGGI) orients the purine to allow stereochemical discrimination between adenine and guanine based on the 6-position. This single interaction is well-conserved in known RRM structures and appears to be a broad indicator for purine preference in the larger family of RRM proteins.

Identification and characterization of the gerH operon of Bacillus anthracis endospores: a differential role for purine nucleosides in germination.

We identified a tri-cistronic operon, gerH, in Bacillus anthracis that is important for endospore germination triggered by two distinct germination response pathways termed inosine-His and purine-Ala. Together, the two pathways allow B. anthracis endospores a broader recognition of purines and amino acids that may be important for host-mediated germination.

Adenine-Aptamer Complexes: A BIPARTITE RNA SITE THAT BINDS THE ADENINE NUCLEIC BASE

RNA aptamers that are able to complex free adenine have been isolated by a SELEX (systematic evolution of ligands by exponential enrichment) procedure. The adenine binding site was revealed by sequence alignment for a prevalent cluster of aptamers, and its structure and interactions with adenine were probed by RNase digestion studies, lead cleavage, boundary determination experiments, and truncated sequences studies. A new purine binding motif was functionally and structurally characterized and compared with other RNAs specific to purine or adenylated compounds. The affinity for adenine and the specificity for other related targets were quantified. This work suggests that the adenine binding site is composed of two independent secondary structure elements forming a bipartite binding site that interacts with adenine in a new mode of purine recognition. Such binding is of great interest because the imidazole moiety is not trapped in the binding site, and would easily be available for catalytic activity.

Amino acid residues N450 and Q449 are critical for the uptake capacity and specificity of UapA, a prototype of a nucleobase-ascorbate transporter family.

Specific carrier-mediated transport of purine and pyrimidine nucleobases across cell membranes is a basic biological process in both prokaryotes and eukaryotes. Recent in silico analysis has shown that the Aspergillus nidulans (UapA, UapC) and bacterial (PbuX, UraA, PyrP) nucleobase transporters, and a group of mammalian L-ascorbic acid transporters (SVCT1 and SVCT2), constitute a unique protein family which includes putative homologues from archea, bacteria, plants and metazoans. The construction and functional analysis of chimeric purine transporters (UapA-UapC) and UapA-specific missense mutations in A. nidulans has previously shown that the region including amino acid residues 378-446 in UapA is critical for purine recognition and transport. Here, we extend our studies on UapA structure-function relationships by studying missense mutations constructed within a 'signature' sequence motif [(F/Y/S)X(Q/E/P)NXGXXXXT(K/R/G)] which is conserved in the putative functional region of all members of the nucleobase/ascorbate transporter family. Residues Q449 and N450 were found to be critical for purine recognition and transport. The results suggest that these residues might directly or indirectly be involved in specific interactions with the purine ring. In particular, interaction of residue 449 with C-2 groups of purines might act as a critical molecular filter involved in the selection of transported substrates. The present and previous mutagenic analyses in UapA suggest that specific polar or charged amino acid residues on either side of an amphipathic alpha-helical transmembrane segment are critical for purine binding and transport.

Chimeric purine transporters of Aspergillus nidulans define a domain critical for function and specificity conserved in bacterial, plant and metazoan homologues.

In Aspergillus nidulans, purine uptake is mediated by three transporter proteins: UapA, UapC and AzgA. UapA and UapC have partially overlapping functions, are 62% identical and have nearly identical predicted topologies. Their structural similarity is associated with overlapping substrate specificities; UapA is a high-affinity, high-capacity specific xanthine/uric acid transporter. UapC is a low/moderate-capacity general purine transporter. We constructed and characterized UapA/UapC, UapC/UapA and UapA/UapC/UapA chimeric proteins and UapA point mutations. The region including residues 378-446 in UapA (336-404 in UapC) has been shown to be critical for purine recognition and transport. Within this region, we identified: (i) one amino acid residue (A404) important for transporter function but probably not for specificity and two residues (E412 and R414) important for UapA function and specificity; and (ii) a sequence, (F/Y/S)X(Q/E/P) NXGXXXXT(K/R/G), which is highly conserved in all homologues of nucleobase transporters from bacteria to man. The UapC/UapA series of chimeras behaves in a linear pattern and leads to an univocal assignment of functional domains while the analysis of the reciprocal and 'sandwich' chimeras revealed unexpected inter-domain interactions. cDNAs coding for transporters including the specificity region defined by these studies have been identified for the first time in the human and Caenorhabditis elegans databases.

Structural features that determine the enzymatic potency and specificity of human angiogenin: threonine-80 and residues 58-70 and 116-123.

Human angiogenin (Ang), a homologue of bovine pancreatic ribonuclease A (RNase A), is a potent inducer of blood vessel formation. It exerts a ribonucleolytic activity that is 10(5)-10(6)-fold lower than that of RNase A but nonetheless essential for biological action. Previous studies revealed some of the structural features of Ang that underlie its catalytic inefficiency: Gln-117 blocks the space corresponding to the pyrimidine binding site of RNase A and Ang lacks the disulfide loop 65-72 that forms most of the purine binding site of RNase A. Additional features have now been identified by mutagenesis and kinetics. Thr-80, which hydrogen-bonds to the pyrimidine-binding residue Thr-44, plays an important part in attenuating activity and in determining pyrimidine specificity: mutation to Ala increases activity toward cytidylyl substrates by 11-15-fold but has only a minimal effect on cleavage of uridylyl substrates. The properties of T44A/T80A and Q117A/T80A double mutants demonstrate that these changes are mediated by Thr-44 and are largely independent of the blockage by Gln-117. The side chain of Ser-118 also suppresses enzymatic activity: S118A is 5-7-fold more effective than Ang. This increase appears to reflect the loss of a hydrogen bond with Asp-116 that helps to orient Gln-117. The effects of deleting residues 119-123 suggest that main-chain atoms of the C-terminal 3(10) helix make a small further contribution. Finally, the significance of the absence of the RNase A loop 65-72 from Ang has been investigated by reexamining the earlier derivative ARH-I (in which Ang residues 58-70 have been replaced by residues 59-73 of RNase) and generating new derivatives of this hybrid protein. The results suggest that the RNase A segment of ARH-I not only provides more effective purine recognition but also counteracts the deleterious effects of Gln-117 and Thr-80 on the pyrimidine site.

An RNA aptamer to the xanthine/guanine base with a distinctive mode of purine recognition.

RNAs that bind to xanthine (2,6-dioxypurine) were isolated from a population of 10(12) random sequences by in vitro selection. These xanthine-binding RNAs were found to have a 10 nt consensus sequence at an internal loop in the most probable secondary structure. By trimming one of the xanthine-binding RNAs, a representative xanthine-binding RNA (designated as XBA) of 32 nt residues was prepared. The dissociation constant of this RNA for xanthine was determined to be 3.3 microM by equilibrium filtration experiments. The XBA RNA can bind to guanine as well, whereas it hardly accommodates adenine, cytosine or uracil. The K d values for various xanthine/guanine analogues were determined, and revealed that the N1H, N7 and O6 moieties of the ligand are involved in the binding with the XBA RNA. The ribonuclease sensitivities of some internal-loop residues changed upon the addition of xanthine, suggesting that the internal loop of the XBA RNA is involved in the ligand binding. Interestingly, the consensus sequence of the xanthine/guanine-binding RNAs is the same as a sequence in one of the internal loops of the hairpin ribozyme, except for a substitution that is neutral with respect to xanthine/guanine binding.

Recognition of the four Watson-Crick base pairs in the DNA minor groove by synthetic ligands.

The design of synthetic ligands that read the information stored in the DNA double helix has been a long-standing goal at the interface of chemistry and biology. Cell-permeable small molecules that target predetermined DNA sequences offer a potential approach for the regulation of gene expression. Oligodeoxynucleotides that recognize the major groove of double-helical DNA via triple-helix formation bind to a broad range of sequences with high affinity and specificity. Although oligonucleotides and their analogues have been shown to interfere with gene expression, the triple-helix approach is limited to recognition of purines and suffers from poor cellular uptake. The subsequent development of pairing rules for minor-groove binding polyamides containing pyrrole (Py) and imidazole (Im) amino acids offers a second code to control sequence specificity. An Im/Py pair distinguishes G x C from C x G and both of these from A x T/T x A base pairs. A Py/Py pair specifies A,T from G,C but does not distinguish AT from T x A. To break this degeneracy, we have added a new aromatic amino acid, 3-hydroxypyrrole (Hp), to the repertoire to test for pairings that discriminate A x T from T x A. We find that replacement of a single hydrogen atom with a hydroxy group in a Hp/Py pairing regulates affinity and specificity by an order of magnitude. By incorporation of this third amino acid, hydroxypyrrole-imidazole-pyrrole polyamides form four ring-pairings (Im/Py, Py/Im, Hp/Py and Py/Hp) which distinguish all four Watson-Crick base pairs in the minor groove of DNA.

Elements of a DNA-polypeptide recognition code: Electrostatic potential around the double helix, and a stereospecific model for purine recognition

A model for stereospecific complex between a polynucleotide double helix and a twisted β-ribbon type polypeptide is described. The β ribbon lies in the major groove with alternate side chains pointing toward the interior of the groove. The base-amino acid complementarities are: Arg, G and Asn or Gln, A. It is shown that this complex can: (a) distinguish between G and A in homopolar sequences; (b) the complex is stabilized by approx. 6 Kcal/mole per hydrogen bond per base pair; (c) the required backbone conformation is in the permitted range of Ramachandran plots.