Structural insights into the morpholino nucleic acid/RNA duplex using the new XNA builder Ducque in a molecular modeling pipeline.

The field of synthetic nucleic acids with novel backbone structures [xenobiotic nucleic acids (XNAs)] has flourished due to the increased importance of XNA antisense oligonucleotides and aptamers in medicine, as well as the development of XNA processing enzymes and new XNA genetic materials. Molecular modeling on XNA structures can accelerate rational design in the field of XNAs as it contributes in understanding and predicting how changes in the sugar-phosphate backbone impact on the complementation properties of the nucleic acids. To support the development of novel XNA polymers, we present a first-in-class open-source program (Ducque) to build duplexes of nucleic acid analogs with customizable chemistry. A detailed procedure is described to extend the Ducque library with new user-defined XNA fragments using quantum mechanics (QM) and to generate QM-based force field parameters for molecular dynamics simulations within standard packages such as AMBER. The tool was used within a molecular modeling workflow to accurately reproduce a selection of experimental structures for nucleic acid duplexes with ribose-based as well as non-ribose-based nucleosides. Additionally, it was challenged to build duplexes of morpholino nucleic acids bound to complementary RNA sequences.

Characterization of an HNA aptamer suggests a non-canonical G-quadruplex motif.

Nucleic acids not only form the basis of heredity, but are increasingly a source of novel nano-structures, -devices and drugs. This has spurred the development of chemically modified alternatives (xeno nucleic acids (XNAs)) comprising chemical configurations not found in nature to extend their chemical and functional scope. XNAs can be evolved into ligands (XNA aptamers) that bind their targets with high affinity and specificity. However, detailed investigations into structural and functional aspects of XNA aptamers have been limited. Here we describe a detailed structure-function analysis of LYS-S8-19, a 1',5'-anhydrohexitol nucleic acid (HNA) aptamer to hen egg-white lysozyme (HEL). Mapping of the aptamer interaction interface with its cognate HEL target antigen revealed interaction epitopes, affinities, kinetics and hot-spots of binding energy similar to protein ligands such as anti-HEL-nanobodies. Truncation analysis and molecular dynamics (MD) simulations suggest that the HNA aptamer core motif folds into a novel and not previously observed HNA tertiary structure, comprising non-canonical hT-hA-hT/hT-hT-hT triplet and hG4-quadruplex structures, consistent with its recognition by two different G4-specific antibodies.

Efficient and Accurate Potential Energy Surfaces of Puckering in Sugar-Modified Nucleosides.

Puckering of the sugar unit in nucleosides and nucleotides is an important structural aspect that directly influences the helical structure of nucleic acids. The preference for specific puckering modes in nucleic acids can be analyzed via in silico conformational analysis, but the large amount of conformations and the accuracy of the analysis leads to an extensive amount of computational time. In this paper, we show that the combination of geometry optimizations with the HF-3c method with single point energies at the RI-MP2 level results in accurate results for the puckering potential energy surface (PES) of DNA and RNA nucleosides while significantly reducing the necessary computational time. Applying this method to a series of known xeno nucleic acids (XNAs) allowed us to rapidly explore the puckering PES of each of the respective nucleosides and to explore the puckering PES of six-membered modified XNA (HNA and ß-homo-DNA) for the first time.

Stable Hairpin Structures Formed by Xylose-Based Nucleic Acids.

Xenobiology explores synthetic nucleic acid polymers as alternative carriers of genetic information to expand the central dogma. The xylo- and deoxyxylo-nucleic acids (XyNA and dXyNA), containing 3' epimers of riboses and deoxyriboses, are considered to be potential candidates for an orthogonal system. In this study, thermal and spectroscopic analyses show that XyNA and dXyNA form stable hairpins. The dXyNA hairpin structure determined by NMR spectroscopy contains a flexible loop that locks the stem into a stable ladder-like duplex with marginal right-handed helicity. The reduced flexibility of the dXyNA duplex observed in the stem of the hairpin demonstrates that folding of dXyNA yields more stable structures described so far.

Phenyltriazole-functionalized sulfamate inhibitors targeting tyrosyl- or isoleucyl-tRNA synthetase.

Antimicrobial resistance is considered as one of the major threats for the near future as the lack of effective treatments for various infections would cause more deaths than cancer by 2050. The development of new antibacterial drugs is considered as one of the cornerstones to tackle this problem. Aminoacyl-tRNA synthetases (aaRSs) are regarded as good targets to establish new therapies. Apart from being essential for cell viability, they are clinically validated. Indeed, mupirocin, an isoleucyl-tRNA synthetase (IleRS) inhibitor, is already commercially available as a topical treatment for MRSA infections. Unfortunately, resistance developed soon after its introduction on the market, hampering its clinical use. Therefore, there is an urgent need for new cellular targets or improved therapies. Follow-up research by Cubist Pharmaceuticals led to a series of selective and in vivo active aminoacyl-sulfamoyl aryltetrazole inhibitors targeting IleRS (e.g. CB 168). Here, we describe the synthesis of new IleRS and TyrRS inhibitors based on the Cubist Pharmaceuticals compounds, whereby the central ribose was substituted for a tetrahydropyran ring. Various linkers were evaluated connecting the six-membered ring with the base-mimicking part of the synthesized analogues. Out of eight novel molecules, a three-atom spacer to the phenyltriazole moiety, which was established using azide-alkyne click chemistry, appeared to be the optimized linker to inhibit IleRS. However, 11 (Ki,app = 88 ± 5.3 nM) and 36a (Ki,app = 114 ± 13.5 nM) did not reach the same level of inhibitory activity as for the known high-affinity natural adenylate-intermediate analogue isoleucyl-sulfamoyl adenosine (IleSA, CB 138; Ki,app = 1.9 ± 4.0 nM) and CB 168, which exhibit a comparable inhibitory activity as the native ligand. Therefore, 11 was docked into the active site of IleRS using a known crystal structure of T. thermophilus in complex with mupirocin. Here, we observed the loss of the crucial 3'- and 4'- hydroxyl group interactions with the target enzyme compared to CB 168 and mupirocin, which we suggest to be the reason for the limited decrease in enzyme affinity. Despite the lack of antibacterial activity, we believe that structurally optimizing these novel analogues via a structure-based approach could ultimately result in aaRS inhibitors which would help to tackle the antibiotic resistance problem.

An N-terminal Ca2+-binding motif regulates the secretory pathway Ca2+/Mn2+-transport ATPase SPCA1.

The Ca2+/Mn2+ transport ATPases 1a and 2 (SPCA1a/2) are closely related to the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and are implicated in breast cancer and Hailey-Hailey skin disease. Here, we purified the human SPCA1a/2 isoforms from a yeast recombinant expression system and compared their biochemical properties after reconstitution. We observed that the purified SPCA1a displays a lower Ca2+ affinity and slightly lower Mn2+ affinity than SPCA2. Remarkably, the turnover rates of SPCA1a in the presence of Mn2+ and SPCA2 incubated with Ca2+ and Mn2+ were comparable, whereas the turnover rate of SPCA1a in Ca2+ was 2-fold higher. Moreover, we noted an unusual biphasic activation curve for the SPCA1a ATPase and autophosphorylation activity, not observed with SPCA2. We also found that the biphasic pattern and low apparent ion affinity of SPCA1a critically depends on ATP concentration. We further show that the specific properties of SPCA1a at least partially depend on an N-terminal EF-hand-like motif, which is present only in the SPCA1a isoform and absent in SPCA2. This motif binds Ca2+, and its mutation lowered the Ca2+ turnover rate relative to that of Mn2+, increased substrate affinity, and reduced the level of biphasic activation of SPCA1a. A biochemical analysis indicated that Ca2+ binding to the N-terminal EF-hand-like motif promotes the activity of SPCA1a by facilitating autophosphorylation. We propose that this regulation may be physiologically relevant in cells with a high Ca2+ load, such as mammary gland cells during lactation, or in cells with a low ATP content, such as keratinocytes.