In Silico Design of a Chimeric Humanized L-asparaginase.

Acute lymphoblastic leukemia (ALL) is the most common cancer among children worldwide, characterized by an overproduction of undifferentiated lymphoblasts in the bone marrow. The treatment of choice for this disease is the enzyme L-asparaginase (ASNase) from bacterial sources. ASNase hydrolyzes circulating L-asparagine in plasma, leading to starvation of leukemic cells. The ASNase formulations of E. coli and E. chrysanthemi present notorious adverse effects, especially the immunogenicity they generate, which undermine both their effectiveness as drugs and patient safety. In this study, we developed a humanized chimeric enzyme from E. coli L-asparaginase which would reduce the immunological problems associated with current L-asparaginase therapy. For these, the immunogenic epitopes of E. coli L-asparaginase (PDB: 3ECA) were determined and replaced with those of the less immunogenic Homo sapiens asparaginase (PDB:4O0H). The structures were modeled using the Pymol software and the chimeric enzyme was modeled using the SWISS-MODEL service. A humanized chimeric enzyme with four subunits similar to the template structure was obtained, and the presence of asparaginase enzymatic activity was predicted by protein-ligand docking.

Inverse Electron-Demand Diels-Alder Bioconjugation Reactions Using 7-Oxanorbornenes as Dienophiles.

Oligonucleotides, peptides, and peptide nucleic acids incorporating 7-oxanorbornene as a dienophile were reacted with tetrazines linked to either a peptide, d-biotin, BODIPY, or N-acetyl-d-galactosamine. The inverse electron-demand Diels-Alder (IEDDA) cycloaddition, which was performed overnight at 37 °C, in all cases furnished the target conjugate in good yields. IEDDA reactions with 7-oxanorbornenes produce a lower number of stereoisomers than that of IEDDA cycloadditions with other dienophiles.

Retro-1-Oligonucleotide Conjugates. Synthesis and Biological Evaluation.

Addition of small molecule Retro-1 has been described to enhance antisense and splice switching oligonucleotides. With the aim of assessing the effect of covalently linking Retro-1 to the biologically active oligonucleotide, three different derivatives of Retro-1 were prepared that incorporated a phosphoramidite group, a thiol or a 1,3-diene, respectively. Retro-1⁻oligonucleotide conjugates were assembled both on-resin (coupling of the phosphoramidite) and from reactions in solution (Michael-type thiol-maleimide reaction and Diels-Alder cycloaddition). Splice switching assays with the resulting conjugates showed that they were active but that they provided little advantage over the unconjugated oligonucleotide in the well-known HeLa Luc705 reporter system.

Compatibility between the cysteine-cyclopentenedione reaction and the copper(i)-catalyzed azide-alkyne cycloaddition.

The cysteine-cyclopentenedione reaction can be combined with the copper(i)-catalyzed azide-alkyne cycloaddition provided that the former is carried out first. If not, the azide and the cyclopentenedione undergo a 1,3-dipolar cycloaddition, which furnishes triazole-containing compounds and products resulting from nitrogen loss. Both of these products were fully characterized. Attempts to obtain either of them as the main compound or to drive the reaction nearly to completion were unsuccessful, which points to the azide-cyclopentenedione reaction as not being useful for bioconjugation.

RNA recognition and self-association of CPEB4 is mediated by its tandem RRM domains.

Cytoplasmic polyadenylation is regulated by the interaction of the cytoplasmic polyadenylation element binding proteins (CPEB) with cytoplasmic polyadenylation element (CPE) containing mRNAs. The CPEB family comprises four paralogs, CPEB1-4, each composed of a variable N-terminal region, two RNA recognition motif (RRM) and a C-terminal ZZ-domain. We have characterized the RRM domains of CPEB4 and their binding properties using a combination of biochemical, biophysical and NMR techniques. Isothermal titration calorimetry, NMR and electrophoretic mobility shift assay experiments demonstrate that both the RRM domains are required for an optimal CPE interaction and the presence of either one or two adenosines in the two most commonly used consensus CPE motifs has little effect on the affinity of the interaction. Both the single RRM1 and the tandem RRM1-RRM2 have the ability to dimerize, although representing a minor population. Self-association does not affect the proteins' ability to interact with RNA as demonstrated by ion mobility-mass spectrometry. Chemical shift effects measured by NMR of the apo forms of the RRM1-RRM2 samples indicate that the two domains are orientated toward each other. NMR titration experiments show that residues on the ß-sheet surface on RRM1 and at the C-terminus of RRM2 are affected upon RNA binding. We propose a model of the CPEB4 RRM1-RRM2-CPE complex that illustrates the experimental data.

On-Resin Conjugation of Diene-Polyamides and Maleimides via Diels-Alder Cycloaddition.

The reaction between maleimides and resin-linked diene-polyamides allows the latter to be used in the preparation of conjugates. Conjugation takes place by reacting the insoluble, hydrophobic diene component either with water-soluble dienophiles or with dienophiles requiring mixtures of water and organic solvents. Experimental conditions can be adjusted to furnish the target conjugate in good yield with no need of adding large excesses of soluble reagent. In case protected maleimides are used, maleimide deprotection and Diels-Alder cycloaddition can be simultaneously carried out to render conjugates with different linking positions. On-resin conjugation is followed by an acidic treatment that removes the polyamide protecting groups with no harm to the cycloadduct, in contrast with the unreacted diene that is indeed degraded under these conditions. Cycloadducts incorporating suitable functional groups can undergo subsequent additional conjugation reactions in solution to furnish double conjugates.

Local RNA flexibility perturbation of the IRES element induced by a novel ligand inhibits viral RNA translation.

The internal ribosome entry site (IRES) element located at the 5'untranslated genomic region of various RNA viruses mediates cap-independent initiation of translation. Picornavirus IRES activity is highly dependent on both its structural organization and its interaction with host factors. Small molecules able to interfere with RNA function are valuable candidates for antiviral agents. Here we show that a small molecule based on benzimidazole (IRAB) inhibited foot-and-mouth disease virus (FMDV) IRES-dependent protein synthesis in cells transfected with infectious RNA leading to a decrease of the virus titer, which was higher than that induced by a structurally related benzimidazole derivative. Interestingly, IRAB preferentially inhibited IRES-dependent translation in cell free systems in a dose-dependent manner. RNA structural analysis by SHAPE demonstrated an increased local flexibility of the IRES structure upon incubation with IRAB, which affected 3 stem-loops (SL) of domain 3. Fluorescence binding assays conducted with individual aminopurine-labeled oligoribonucleotides indicated that the SL3A binds IRAB (EC50 18 µM). Taken together, the results derived from SHAPE reactivity and fluorescence binding assays suggested that the target site of IRAB within the FMDV IRES might be a folded RNA structure that involves the entire apical region of domain 3. Our data suggest that the conformational changes induced by this compound on a specific region of the IRES structure which is essential for its activity is, at least in part, responsible for the reduced IRES efficiency observed in cell free lysates and, particularly, in RNA-transfected cells.

Exploiting protected maleimides to modify oligonucleotides, peptides and peptide nucleic acids.

This manuscript reviews the possibilities offered by 2,5-dimethylfuran-protected maleimides. Suitably derivatized building blocks incorporating the exo Diels-Alder cycloadduct can be introduced at any position of oligonucleotides, peptide nucleic acids, peptides and peptoids, making use of standard solid-phase procedures. Maleimide deprotection takes place upon heating, which can be followed by either Michael-type or Diels-Alder click conjugation reactions. However, the one-pot procedure in which maleimide deprotection and conjugation are simultaneously carried out provides the target conjugate more quickly and, more importantly, in better yield. This procedure is compatible with conjugates involving oligonucleotides, peptides and peptide nucleic acids. A variety of cyclic peptides and oligonucleotides can be obtained from peptide and oligonucleotide precursors incorporating protected maleimides and thiols.

Orthogonal protection of peptides and peptoids for cyclization by the thiol-ene reaction and conjugation.

Cyclic peptides and peptoids were prepared using the thiol-ene Michael-type reaction. The linear precursors were provided with additional functional groups allowing for subsequent conjugation: an orthogonally protected thiol, a protected maleimide, or an alkyne. The functional group for conjugation was placed either within the cycle or in an external position. The click reactions employed for conjugation with suitably derivatized nucleoside or oligonucleotides were either cycloadditions (Diels-Alder, Cu(I)-catalyzed azide-alkyne) or the same Michael-type reaction as for cyclization.

A minimal i-motif stabilized by minor groove G:T:G:T tetrads.

The repetitive DNA sequences found at telomeres and centromeres play a crucial role in the structure and function of eukaryotic chromosomes. This role may be related to the tendency observed in many repetitive DNAs to adopt non-canonical structures. Although there is an increasing recognition of the importance of DNA quadruplexes in chromosome biology, the co-existence of different quadruplex-forming elements in the same DNA structure is still a matter of debate. Here we report the structural study of the oligonucleotide d(TCGTTTCGT) and its cyclic analog d. Both sequences form dimeric quadruplex structures consisting of a minimal i-motif capped, at both ends, by a slipped minor groove-aligned G:T:G:T tetrad. These mini i-motifs, which do not exhibit the characteristic CD spectra of other i-motif structures, can be observed at neutral pH, although they are more stable under acidic conditions. This finding is particularly relevant since these oligonucleotide sequences do not contain contiguous cytosines. Importantly, these structures resemble the loop moiety adopted by an 11-nucleotide fragment of the conserved centromeric protein B (CENP-B) box motif, which is the binding site for the CENP-B.

Protected maleimide building blocks for the decoration of peptides, peptoids, and peptide nucleic acids.

Monomers allowing for the introduction of [2,5-dimethylfuran]-protected maleimides into polyamides such as peptides, peptide nucleic acids, and peptoids were prepared, as well as the corresponding oligomers. Suitable maleimide deprotection conditions were established in each case. The stability of the adducts generated by Michael-type maleimide-thiol reaction and Diels-Alder cycloaddition to maleimide deprotection conditions was exploited to prepare a variety of conjugates from peptide and PNA scaffolds incorporating one free and one protected maleimide. The target molecules were synthesized by using two subsequent maleimide-involving click reactions separated by a maleimide deprotection step. Carrying out maleimide deprotection and conjugation simultaneously gave better results than performing the two reactions subsequently.

The effect of loop residues in four-stranded dimeric structures stabilized by minor groove tetrads.

Some DNA oligonucleotides can fold back and self-associate forming dimeric structures stabilized by intermolecular base pairs. The resulting antiparallel dimer is a tightly packed four-stranded structure formed by a core of minor groove tetrads connected by short loops of unpaired nucleotides. We have explored the sequential requirements for the loop residues and have found that this family of structures is only stable with one- and two-residue loops, with the stability of the former ones being only marginal. Two-residue loops with purines in the first position give rise to the most stable structures due to their enhanced stacking interaction with the adjacent minor groove tetrad. On the other hand, pyrimidines confer more stability than purines in the second position of the loop.

Conjugation reactions involving maleimides and phosphorothioate oligonucleotides.

Phosphorothioate diester oligonucleotides proved to be fully compatible with maleimides in the context of two different conjugation reactions: (a) reaction of (5')diene-[phosphorothioate oligonucleotides] with maleimido-containing compounds to afford the Diels-Alder cycloadduct; (b) conjugation of (5')maleimido-[phosphorothioate oligonucleotides] with thiol-containing compounds. No evidence of reaction between phosphorothioate diesters and maleimides was found in any of these processes. Importantly, in the preparation of (5')maleimido-[phosphorothioate oligonucleotides] from [protected maleimido]-[phosphorothioate oligonucleotides], which requires the maleimide to be deprotected by retro-Diels-Alder reaction (heating for 3-4 h in toluene at 90 °C), no addition of phosphorothioate diester to the maleimide was found either. Finally, maleimide-[phosphorothioate monoester] conjugation was also explored for comparison purposes.

Easy introduction of maleimides at different positions of oligonucleotide chains for conjugation purposes.

[2,5-Dimethylfuran]-protected maleimides were placed at both internal positions and the 3'-end of oligonucleotides making use of solid-phase synthesis procedures. A new phosphoramidite derivative and a new solid support incorporating the protected maleimide moiety were prepared for this purpose. In all cases maleimide deprotection (retro-Diels-Alder reaction) followed by reaction with thiol-containing compounds afforded the target conjugate.

Genetic reductionist approach for dissecting individual roles of GGDEF proteins within the c-di-GMP signaling network in Salmonella.

Bacteria have developed an exclusive signal transduction system involving multiple diguanylate cyclase and phosphodiesterase domain-containing proteins (GGDEF and EAL/HD-GYP, respectively) that modulate the levels of the same diffusible molecule, 3'-5'-cyclic diguanylic acid (c-di-GMP), to transmit signals and obtain specific cellular responses. Current knowledge about c-di-GMP signaling has been inferred mainly from the analysis of recombinant bacteria that either lack or overproduce individual members of the pathway, without addressing potential compensatory effects or interferences between them. Here, we dissected c-di-GMP signaling by constructing a Salmonella strain lacking all GGDEF-domain proteins and then producing derivatives, each restoring 1 protein. Our analysis showed that most GGDEF proteins are constitutively expressed and that their expression levels are not interdependent. Complete deletion of genes encoding GGDEF-domain proteins abrogated virulence, motility, long-term survival, and cellulose and fimbriae synthesis. Separate restoration revealed that 4 proteins from Salmonella and 1 from Yersinia pestis exclusively restored cellulose synthesis in a c-di-GMP-dependent manner, indicating that c-di-GMP produced by different GGDEF proteins can activate the same target. However, the restored strain containing the STM4551-encoding gene recovered all other phenotypes by means of gene expression modulation independently of c-di-GMP. Specifically, fimbriae synthesis and virulence were recovered through regulation of csgD and the plasmid-encoded spvAB mRNA levels, respectively. This study provides evidence that the regulation of the GGDEF-domain proteins network occurs at 2 levels: a level that strictly requires c-di-GMP to control enzymatic activities directly, restricted to cellulose synthesis in our experimental conditions, and another that involves gene regulation for which c-di-GMP synthesis can be dispensable.

Self-association of short DNA loops through minor groove C:G:G:C tetrads.

In addition to the better known guanine-quadruplex, four-stranded nucleic acid structures can be formed by tetrads resulting from the association of Watson-Crick base pairs. When such association occurs through the minor groove side of the base pairs, the resulting structure presents distinctive features, clearly different from quadruplex structures containing planar G-tetrads. Although we have found this unusual DNA motif in a number of cyclic oligonucleotides, this is the first time that this DNA motif is found in linear oligonucleotides in solution, demonstrating that cyclization is not required to stabilize minor groove tetrads in solution. In this article, we have determined the solution structure of two linear octamers of sequence d(TGCTTCGT) and d(TCGTTGCT), and their cyclic analogue d, utilizing 2D NMR spectroscopy and restrained molecular dynamics. These three molecules self-associate forming symmetric dimers stabilized by a novel kind of minor groove C:G:G:C tetrad, in which the pattern of hydrogen bonds differs from previously reported ones. We hypothesize that these quadruplex structures can be formed by many different DNA sequences, but its observation in linear oligonucleotides is usually hampered by competing Watson-Crick duplexes.

Putative one-pot prebiotic polypeptides with ribonucleolytic activity.

KIA7, a peptide with a highly restricted set of amino acids (Lys, Ile, Ala, Gly and Tyr), adopts a specifically folded structure. Some amino acids, including Lys, Ile, Ala, Gly and His, form under the same putative prebiotic conditions, whereas different conditions are needed for producing Tyr, Phe and Trp. Herein, we report the 3D structure and conformational stability of the peptide KIA7H, which is composed of only Lys, Ile, Ala, Gly and His. When the imidazole group is neutral, this 20-mer peptide adopts a four-helix bundle with a specifically packed hydrophobic core. Therefore, one-pot prebiotic proteins with well-defined structures might have arisen early in chemical evolution. The Trp variant, KIA7W, was also studied. It adopts a 3D structure similar to that of KIA7H and its previously studied Tyr and Phe variants, but is remarkably more stable. When tested for ribonucleolytic activity, KIA7H, KIA7W and even short, unstructured peptides rich in His and Lys, in combination with Mg(++), Mn(++) or Ni(++) (but not Cu(++), Zn(++) or EDTA) specifically cleave the single-stranded region in an RNA stem-loop. This suggests that prebiotic peptide-divalent cation complexes with ribonucleolytic activity might have co-inhabited the RNA world.

Self-association of cyclic oligonucleotides through G:T:G:T minor groove tetrads.

Minor groove aligned tetrads resulting from the association of Watson-Crick base pairs stabilize a distinct class of four-stranded DNA structures, different from G-quadruplexes or i-motifs. These tetrads can be formed by several arrangements of G-C or A-T base pairs. Here we prove that minor groove tetrads can be also formed by G-T mismatches. In this manuscript we describe the dimeric solution structures of two cyclic oligonucleotides stabilized by intermolecular G-T non-canonical base pairs. In the dimeric structure of d, these mismatches interact to each other giving rise to minor groove aligned G:T:G:T or mixed G:T:G:C tetrads. Interestingly, the stability conferred by mismatched G-T containing tetrads is similar to that of minor groove tetrads solely formed by G-C Watson-Crick base pairs.

Towards a Situated Spatial Epidemiology of Violence: A Placially-Informed Geospatial Analysis of Homicide in Alagoas, Brazil.

This paper presents an empirically grounded call for a more nuanced engagement and situatedness with placial characteristics within a spatial epidemiology frame. By using qualitative data collected through interviews and observation to parameterise standard and spatial regression models, and through a critical interpretation of their results, we present initial inroads for a situated spatial epidemiology and an analytical framework for health/medical geographers to iteratively engage with data, modelling, and the context of both the subject and process of analysis. In this study, we explore the socioeconomic factors that influence homicide rates in the Brazilian state of Alagoas from a critical public health perspective. Informed by field observation and interviews with 24 youths in low-income neighbourhoods and prisons in Alagoas, we derive and critically reflect on three regression models to predict municipal homicide rates from 2016-2020. The model results indicate significant effects for the male population, persons without elementary school completion, households with reported income, divorced persons, households without piped water, and persons working outside their home municipality. These results are situated in the broader socioeconomic context, trajectories, and cycles of inequality in the study area and underscore the need for integrative and contextually engaged mixed method study design in spatial epidemiology.

Diels-Alder cycloadditions in water for the straightforward preparation of peptide-oligonucleotide conjugates.

The Diels-Alder reaction between diene-modified oligonucleotides and maleimide-derivatized peptides afforded peptide-oligonucleotide conjugates with high purity and yield. Synthesis of the reagents was easily accomplished by on-column derivatization of the corresponding peptides and oligonucleotides. The cycloaddition reaction was carried out in mild conditions, in aqueous solution at 37 degrees C. The speed of the reaction was found to vary depending on the size of the reagents, but it can be completed in 8-10 h by reacting the diene-oligonucleotide with a small excess of maleimide-peptide.