Search

Inefficient endosomal escape remains the primary barrier to the broad application of oligonucleotide therapeutics. Liver uptake after systemic administration is sufficiently robust that a therapeutic effect can be achieved but targeting extrahepatic tissues remains challenging. Prior attempts to improve oligonucleotide activity using small molecules that increase the leakiness of endosomes have failed due to unacceptable toxicity. Here, we show that the well-tolerated and orally bioavailable synthetic sphingolipid analog, SH-BC-893, increases the activity of antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) up to 200-fold in vitro without permeabilizing endosomes. SH-BC-893 treatment trapped endocytosed oligonucleotides within extra-lysosomal compartments thought to be more permeable due to frequent membrane fission and fusion events. Simultaneous disruption of ARF6-dependent endocytic recycling and PIKfyve-dependent lysosomal fusion was necessary and sufficient for SH-BC-893 to increase non-lysosomal oligonucleotide levels and enhance their activity. In mice, oral administration of SH-BC-893 increased ASO potency in the liver by 15-fold without toxicity. More importantly, SH-BC-893 enabled target RNA knockdown in the CNS and lungs of mice treated subcutaneously with cholesterol-functionalized duplexed oligonucleotides or unmodified ASOs, respectively. Together, these results establish the feasibility of using a small molecule that disrupts endolysosomal trafficking to improve the activity of oligonucleotides in extrahepatic tissues.

Nucleic acids research / Special publication xx, gkac774 (2022). doi:10.1093/nar/gkac774, The introduction of phosphorothioate (PS) linkages to the backbone of therapeutic nucleic acids substantially increases their stability and potency. It also affects their interactions with cellular proteins, but the molecular mechanisms that underlie this effect are poorly understood. Here, we report structural and biochemical studies of interactions between annexin A2, a protein that does not possess any known canonical DNA binding domains, and phosphorothioate-modified antisense oligonucleotides. We show that a unique mode of hydrophobic interactions between a sulfur atom of the phosphorothioate group and lysine and arginine residues account for the enhanced affinity of modified nucleic acid for the protein. Our results demonstrate that this mechanism of interaction is observed not only for nucleic acid-binding proteins but can also account for the association of PS oligonucleotides with other proteins. Using the anomalous diffraction of sulfur, we showed that preference for phosphorothioate stereoisomers is determined by the hydrophobic environment around the PS linkage that comes not only from protein but also from additional structural features within the ASO such as 5-Me groups on cytosine nucleobases., Published by Information Retrieval Ltd., London

20 pages, 3 tables, 5 figures.-- Samantha Andrzejaczek ... et al.-- Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC), Knowledge of the three-dimensional movement patterns of elasmobranchs is vital to understand their ecological roles and exposure to anthropogenic pressures. To date, comparative studies among species at global scales have mostly focused on horizontal movements. Our study addresses the knowledge gap of vertical movements by compiling the first global synthesis of vertical habitat use by elasmobranchs from data obtained by deployment of 989 biotelemetry tags on 38 elasmobranch species. Elasmobranchs displayed high intra- and interspecific variability in vertical movement patterns. Substantial vertical overlap was observed for many epipelagic elasmobranchs, indicating an increased likelihood to display spatial overlap, biologically interact, and share similar risk to anthropogenic threats that vary on a vertical gradient. We highlight the critical next steps toward incorporating vertical movement into global management and monitoring strategies for elasmobranchs, emphasizing the need to address geographic and taxonomic biases in deployments and to concurrently consider both horizontal and vertical movements, Data analysis was funded by the Bertarelli Foundation through the Marine Science program through grants to D.J.C., B.A.B., and S.A. D.J.C. is also funded through Research England, UK. S.A. and B.A.B. thank the Moore Foundation and the Packard Foundation. F.G.-M. thanks the Instituto Politecnico Nacional for fellowships (COFAA, EDI). S.B.W. thanks funding from the Darwin Initiative (DPLUS046). A.D.M.D. acknowledges funding from the Research and Conservation Budget at Georgia Aquarium, including philanthropic gifts from several anonymous donors. K.F. acknowledges funding from the Rolex Awards for Enterprise and the Whitley Fund for Nature

The Northwest Atlantic (NWA) population of porbeagles Lamna nasus is susceptible to capture in rod-and-reel fisheries and most individuals are discarded alive due to catch and size limits. To estimate post-release survival, pop-off satellite archival tags were attached to porbeagles captured with rod-and-reel. Fourteen tags were deployed, of which 13 transmitted. All sharks for which we had data survived, giving a post-release survival rate of 100%. Following release, 6 individuals remained in surface waters for several hours to days, while 2 individuals immediately resumed normal diving behaviors. For the remaining sharks (n = 5), low tag transmission resolution precluded the detection of fine scale post-release behavior. The duration of initial depth-holding behavior was characterized using a break-point analysis of dive track variance, which suggests porbeagles exhibited a median post-release recovery period of 116 h (10th and 90th percentiles = 68.8 and 280.1 h) following capture and handling. Our preliminary study suggests immature porbeagles are resilient to capture and handling, although more data would provide stronger support for management recommendations.

The cyanuric acid (CA) heterocycle forms supramolecular structures with adenine nucleobases/nucleosides and oligonucleotides, leading to speculation that they can act as forerunners to RNA. Herein, the assembly behavior of RNA containing CA and CA-ribose nucleoside was studied. Contrary to previous reports, CA in RNA and the CA-ribonucleoside resulted in destabilization of supramolecular assemblies, which led to a reevaluation of the CA-adenine hexameric rosette structure. An unprecedented noncovalent supramolecular helicene structure is proposed to account for the striking difference in behavior, which has implications for novel paradigms for reorganizing the structures of nucleic acids, the synthesis of long helicenes, and pre-RNA world paradigms. The results caution against extrapolating the self-assembly behavior of individual heterocycles from the level of monomers to oligomers because the base-paring properties of (non-)canonical nucleobases are impacted by the type of oligomeric backbone to which they are attached.

Previously, we reported the creation of a semi-synthetic organism (SSO) that stores and retrieves increased information by virtue of stably maintaining an unnatural base pair (UBP) in its DNA, transcribing the corresponding unnatural nucleotides into the codons and anticodons of mRNAs and tRNAs, and then using them to produce proteins containing noncanonical amino acids (ncAAs). Here we report a systematic extension of the effort to optimize the SSO by exploring a variety of deoxy- and ribonucleotide analogues. Importantly, this includes the first in vivo structure-activity relationship (SAR) analysis of unnatural ribonucleoside triphosphates. Similarities and differences between how DNA and RNA polymerases recognize the unnatural nucleotides were observed, and remarkably, we found that a wide variety of unnatural ribonucleotides can be efficiently transcribed into RNA and then productively and selectively paired at the ribosome to mediate the synthesis of proteins with ncAAs. The results extend previous studies, demonstrating that nucleotides bearing no significant structural or functional homology to the natural nucleotides can be efficiently and selectively paired during replication, to include each step of the entire process of information storage and retrieval. From a practical perspective, the results identify the most optimal UBP for replication and transcription, as well as the most optimal unnatural ribonucleoside triphosphates for transcription and translation. The optimized SSO is now, for the first time, able to efficiently produce proteins containing multiple, proximal ncAAs.

BACKGROUND AND AIM: Tropical Cyclones (TCs) can cause immense harm to people, so estimating their potential health impacts is an essential part of disaster planning. Potential TC-associated exposur...

We engineered a machine learning approach, MSHub, to enable auto-deconvolution of gas chromatography–mass spectrometry (GC–MS) data. We then designed workflows to enable the community to store, process, share, annotate, compare and perform molecular networking of GC–MS data within the Global Natural Product Social (GNPS) Molecular Networking analysis platform. MSHub/GNPS performs auto-deconvolution of compound fragmentation patterns via unsupervised non-negative matrix factorization and quantifies the reproducibility of fragmentation patterns across samples., Fil: Aksenov, Alexander A. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences; Estados Unidos, Fil: Aksenov, Alexander A. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences. Collaborative Mass Spectrometry Innovation Center; Estados Unidos, Fil: Laponogov, Ivan. South Kensington Campus. Imperial College London. Department of Surgery and Cancer; Reino Unido, Fil: Zhang, Zheng. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences; Estados Unidos, Fil: Doran, Sophie L. F. South Kensington Campus. Imperial College London. Department of Surgery and Cancer; Reino Unido, Fil: Belluomo, Ilaria. South Kensington Campus. Imperial College London. Department of Surgery and Cancer; Reino Unido, Fil: Veselkov, Dennis. Intelligify Limited. Reino Unido. South Kensington Campus. Imperial College. Department of Computing; Reino Unido, Fil: Bittremieux, Wout. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences; Estados Unidos, Fil: Bittremieux, Wout. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences. Collaborative Mass Spectrometry Innovation Center; Estados Unidos, Fil: Bittremieux, Wout. University of Antwerp. Department of Computer Science; Belgica, Fil: Nothias, Louis Felix. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences; Estados Unidos, Fil: Nothias, Louis Felix. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences. Collaborative Mass Spectrometry Innovation Center; Estados Unidos, Fil: Nothias-Esposito, Mélissa. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences; Estados Unidos, Fil: Nothias-Esposito, Mélissa. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences. Collaborative Mass Spectrometry Innovation Center; Estados Unidos, Fil: Maloney, Katherine N. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences; Estados Unidos, Fil: Maloney, Katherine N. Point Loma Nazarene University. Department of Chemistry; Estados Unidos, Fil: Misra, Biswapriya B. Wake Forest School of Medicine. Section of Molecular Medicine. Department of Internal Medicine. Center for Precision Medicine; Estados Unidos, Fil: Melnik, Alexey V. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences; Estados Unidos, Fil: Smirnov, Aleksandr. University of North Carolina at Charlotte. Department of Bioinformatics and Genomics; Estados Unidos, Fil: Du, Xiuxia. University of North Carolina at Charlotte. Department of Bioinformatics and Genomics; Estados Unidos, Fil: Jones, Kenneth L. University of North Carolina at Charlotte. Department of Bioinformatics and Genomics; Estados Unidos, Fil: Dorrestein, Kathleen. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences; Estados Unidos, Fil: Dorrestein, Kathleen. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences. Collaborative Mass Spectrometry Innovation Center; Estados Unidos, Fil: Panitchpakdi, Morgan. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences; Estados Unidos, Fil: Ernst, Madeleine. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences; Estados Unidos, Fil: Ernst, Madeleine. Statens Serum Institut. Danish Center for Neonatal Screening. Department of Congenital Disorders. Section for Clinical Mass Spectrometry; Dinamarca, Fil: van der Hooft, Justin J. J. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences; Estados Unidos, Fil: van der Hooft, Justin J. J. Wageningen University. Bioinformatics Group; Países Bajos, Fil: Gonzalez, Mabel. Universidad de los Andes. Department of Chemistry; Colombia, Fil: Carazzone, Chiara. Universidad de los Andes. Department of Chemistry; Colombia, Fil: Amézquita, Adolfo. Universidad de los Andes. Department of Biological Sciences; Colombia, Fil: Callewaert, Chris. Center for Microbial Ecology and Technology; Belgica, Fil: Callewaert, Chris. University of California. Department of Pediatrics; Estados Unidos, Fil: Morton, James T. University of California. Department of Pediatrics; Estados Unidos, Fil: Morton, James T. Simons Foundation. Flatiron Institute. Center for Computational Biology; Estados Unidos, Fil: Quinn, Robert A. Michigan State University. Department of Biochemistry and Molecular Biology; Estados Unidos, Fil: Bouslimani, Amina. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences; Estados Unidos, Fil: Bouslimani, Amina. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences. Collaborative Mass Spectrometry Innovation Center; Estados Unidos, Fil: Orio, Andrea Albarracín. Universidad Católica de Córdoba. IRNASUS. CONICET. Facultad de Ciencias Agropecuarias; Argentina, Fil: Petras, Daniel. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences; Estados Unidos, Fil: Petras, Daniel. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences. Collaborative Mass Spectrometry Innovation Center; Estados Unidos, Fil: Smania, Andrea M. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica Ranwel Caputto; Argentina, Fil: Smania, Andrea M. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. CONICET; Argentina, Fil: Couvillion, Sneha P. Pacific Northwest National Laboratory. Biological Sciences Division; Estados Unidos, Fil: Burnet, Meagan C. Pacific Northwest National Laboratory. Biological Sciences Division; Estados Unidos, Fil: Nicora, Carrie D. Pacific Northwest National Laboratory. Biological Sciences Division; Estados Unidos, Fil: Zink, Erika. Pacific Northwest National Laboratory. Biological Sciences Division; Estados Unidos, Fil: Metz, Thomas O. Pacific Northwest National Laboratory. Biological Sciences Division; Estados Unidos, Fil: Artaev, Viatcheslav. LECO Corporation; Estados Unidos, Fil: Humston-Fulmer, Elizabeth. LECO Corporation; Estados Unidos, Fil: Gregor, Rachel. Ben-Gurion University of the Negev. Department of Chemistry and the National Institute for Biotechnology in the Negev; Israel, Fil: Meijler, Michael M. Ben-Gurion University of the Negev. Department of Chemistry and the National Institute for Biotechnology in the Negev; Israel, Fil: Mizrahi, Itzhak. Ben-Gurion University of the Negev. Department of Life Sciences and the National Institute for Biotechnology in the Negev; Israel, Fil: Eyal, Stav. Ben-Gurion University of the Negev. Department of Life Sciences and the National Institute for Biotechnology in the Negev; Israel, Fil: Anderson, Brooke. University of California. Division of Biological Sciences; Estados Unidos, Fil: Dutton, Rachel. University of California. Division of Biological Sciences; Estados Unidos, Fil: Lugan, Raphaël. Université d’Avignon et des Pays du Vaucluse, Agrosciences. UMR Qualisud; Francia, Fil: Boulch, Pauline Le. Université d’Avignon et des Pays du Vaucluse, Agrosciences. UMR Qualisud; Francia, Fil: Guitton, Yann. INRAe. Oniris. Laboratoire d’Etude des Résidus et Contaminants dans les Aliments (LABERCA); Francia, Fil: Prevost, Stephanie. INRAe. Oniris. Laboratoire d’Etude des Résidus et Contaminants dans les Aliments (LABERCA); Francia, Fil: Poirier, Audrey. INRAe. Oniris. Laboratoire d’Etude des Résidus et Contaminants dans les Aliments (LABERCA); Francia, Fil: Dervilly, Gaud. INRAe. Oniris. Laboratoire d’Etude des Résidus et Contaminants dans les Aliments (LABERCA); Francia, Fil: Le Bizec, Bruno. Oniris. Laboratoire d’Etude des Résidus et Contaminants dans les Aliments (LABERCA); Francia, Fil: Fait, Aaron. Ben Gurion University of the Negev. Sede Boqer Campus. The Jacob Blaustein Institutes for Desert Research. The French Associates Institute for Agriculture and Biotechnology of Dryland; Israel, Fil: Persi, Noga Sikron. Ben Gurion University of the Negev. Sede Boqer Campus. The Jacob Blaustein Institutes for Desert Research. The French Associates Institute for Agriculture and Biotechnology of Dryland; Israel, Fil: Song, Chao. Ben Gurion University of the Negev. Sede Boqer Campus. The Jacob Blaustein Institutes for Desert Research. The French Associates Institute for Agriculture and Biotechnology of Dryland; Israel, Fil: Gashu, Kelem. Ben Gurion University of the Negev. Sede Boqer Campus. The Jacob Blaustein Institutes for Desert Research. The French Associates Institute for Agriculture and Biotechnology of Dryland; Israel, Fil: Coras, Roxana. University of California. Department of Medicine. Division of Rheumatology; Estados Unidos, Fil: Guma, Monica. University of California. Department of Medicine. Division of Rheumatology; Estados Unidos, Fil: Manasson, Julia. New York University School of Medicine. Department of Medicine. Division of Rheumatology; Estados Unidos, Fil: Scher, Jose U. New York University School of Medicine. Department of Medicine. Division of Rheumatology; Estados Unidos, Fil: Barupal, Dinesh Kumar. Icahn School of Medicine at Mount Sinai. Department of Environmental Medicine and Public Health; Estados Unidos, Fil: Alseekh, Saleh. Max Planck Institute for Molecular Plant Physiology; Alemania, Fil: Alseekh, Saleh. Center of Plant Systems Biology and Biotechnology (CPSBB); Bulgaria, Fil: Fernie, Alisdair R. Max Planck Institute for Molecular Plant Physiology; Alemania, Fil: Fernie, Alisdair R. Center of Plant Systems Biology and Biotechnology (CPSBB); Bulgaria, Fil: Mirnezami, Reza. Royal Free Hospital NHS Foundation Trust. Department of Colorectal Surgery; Reino Unido, Fil: Vasiliou, Vasilis. Yale University. Yale School of Public Health. Department of Environmental Health Sciences; Estados Unidos, Fil: Schmid, Robin. University of Münster. Institute of Inorganic and Analytical Chemistry; Alemania, Fil: Borisov, Roman S. A.V. Topchiev Institute of Petrochemical Synthesis RAS; Rusia, Fil: Kulikova, Larisa N. Рeoples’ Friendship University of Russia (RUDN University); Rusia, Fil: Knight, Rob. University of California. Department of Pediatrics; Estados Unidos, Fil: Knight, Rob. University of California. UCSD Center for Microbiome Innovation; Estados Unidos, Fil: Knight, Rob. University of California. Department of Bioengineering; Estados Unidos, Fil: Wang, Mingxun. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences; Estados Unidos, Fil: Wang, Mingxun. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences. Collaborative Mass Spectrometry Innovation Center; Estados Unidos, Fil: Hanna, George B. South Kensington Campus. Imperial College London. Department of Surgery and Cancer; Reino Unido, Fil: Dorrestein, Pieter C. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences; Estados Unidos, Fil: Dorrestein, Pieter C. University of California. Skaggs School of Pharmacy and Pharmaceutical Sciences. Collaborative Mass Spectrometry Innovation Center; Estados Unidos, Fil: Dorrestein, Pieter C. University of California. Department of Pediatrics; Estados Unidos, Fil: Veselkov, Kirill. South Kensington Campus. Imperial College London. Department of Surgery and Cancer; Reino Unido

Filamentous marine cyanobacteria make a variety of bioactive molecules that are produced by polyketide synthases, non-ribosomal peptide synthetases, and hybrid pathways that are encoded by large biosynthetic gene clusters. These cyanobacterial natural products represent potential drugs leads; however, thorough pharmacological investigations have been impeded by the limited quantity of compound that is typically available from the native organisms. Additionally, investigations of the biosynthetic gene clusters and enzymatic pathways have been difficult due to the inability to conduct genetic manipulations in the native producers. Here we report a set of genetic tools for the heterologous expression of biosynthetic gene clusters in the cyanobacteria Synechococcus elongatus PCC 7942 and Anabaena (Nostoc) PCC 7120. To facilitate the transfer of gene clusters in both strains, we engineered a strain of Anabaena that contains S. elongatus homologous sequences for chromosomal recombination at a neutral site and devised a CRISPR-based strategy to efficiently obtain segregated double recombinant clones of Anabaena. These genetic tools were used to express the large 28.7 kb cryptomaldamide biosynthetic gene cluster from the marine cyanobacterium Moorena (Moorea) producens JHB in both model strains. S. elongatus did not produce cryptomaldamide, however high-titer production of cryptomaldamide was obtained in Anabaena. The methods developed in this study will facilitate the heterologous expression of biosynthetic gene clusters isolated from marine cyanobacteria and complex metagenomic samples.Abstract Figure

The phosphorothioate backbone modification (PS) is one of the most widely used chemical modifications for enhancing the drug-like properties of nucleic acid-based drugs, including antisense oligonucleotides (ASOs). PS-modified nucleic acid therapeutics show improved metabolic stability from nuclease-mediated degradation and exhibit enhanced interactions with plasma, cell-surface, and intracellular proteins, which facilitates their tissue distribution and cellular uptake in animals. However, little is known about the structural basis of the interactions of PS nucleic acids with proteins. Here, we report a crystal structure of the DNA-binding domain of a model ASO-binding protein PC4, in complex with a full PS 2'-OMe DNA gapmer ASO. To our knowledge this is the first structure of a complex between a protein and fully PS nucleic acid. Each PC4 dimer comprises two DNA-binding interfaces. In the structure one interface binds the 5'-terminal 2'-OMe PS flank of the ASO, while the other interface binds the regular PS DNA central part in the opposite polarity. As a result, the ASO forms a hairpin-like structure. ASO binding also induces the formation of a dimer of dimers of PC4, which is stabilized by base pairing between homologous regions of the ASOs bound by each dimer of PC4. The protein interacts with the PS nucleic acid through a network of electrostatic and hydrophobic interactions, which provides insights into the origins for the enhanced affinity of PS for proteins. The importance of these contacts was further confirmed in a NanoBRET binding assay using a Nano luciferase tagged PC4 acting as the BRET donor, to a fluorescently conjugated ASO acting as the BRET acceptor. Overall, our results provide insights into the molecular forces that govern the interactions of PS ASOs with cellular proteins and provide a potential model for how these interactions can template protein-protein interactions causative of cellular toxicity.

Pyrophosphate linkages are important in extant biology and are hypothesized to have played a role in prebiotic chemistry and in the origination of oligonucleotides. Inspired by pyrophosphate as backbones of primordial oligomers, DNA oligomers with varying amounts of pyrophosphate inserts (ppDNA) were synthesized and investigated for their base-pairing properties. As expected, pyrophosphate inserts into the backbone compromised the thermal stability of ppDNA-DNA duplexes. In contrast, the ppDNA-RNA duplex exhibited, remarkably, duplex stability, even with accumulation of pyrophosphate linkages. This seems to be a consequence of an increase in the diameter of the double-helix with eight-bond-repeat units, and higher inclination of the base-pair axis with respect to the backbone in RNA (A-form), compared with that in DNA (B-form). These results suggest that pyrophosphate-linked oligonucleotides could harbor functional capabilities with implications for their roles in the origins of life and chemical biology.

This study evaluated the effect of replacing soybean meal (SBM) with DDGS and crystalline amino acids on growth and carcass lean. Pigs (n = 512; 38.51±0.13 kg BW) were blocked by BW and sex and placed in 64 pens (4 gilts and barrows per pen). Treatments were arranged as a 2×4 factorial with DDGS included at 0 or 25% and L-lysine-HCl (LYS) added at 0, 0.2, 0.4, and 0.6%. Dietary SBM inclusion declined as LYS increased from 32.06 to 13.14% (Phase 1) and 28.25 to 9.40% (Phase 2) for control diets. It declined from 27.85 to 8.89% (Phase 1) and 24.05 to 5.10% (Phase 2) for DDGS diets. Diets contained 1.00 (Phase 1, 21 days) and 0.90% (Phase 2, 18 days) SID lysine and were balanced for ideal protein and net energy. During Phase 1, DDGS decreased ADG (P = 0.06; 786 vs. 821 g/d). Increasing LYS increased (quadratic, P ≤ 0.05) ADG and ADFI with the greatest response at 0.4% LYS. G:F decreased (linear, P = 0.035) with increasing LYS. During Phase 2, increasing LYS in control, but not DDGS diets, decreased (linear, P < 0.005) ADG and ADFI. G:F declined (P = 0.054) with DDGS inclusion (370 vs. 383 g/kg). Overall, ADG decreased (linear, P = 0.005) as LYS increased in control (959, 929, 908, 860 g/d), but not DDGS diets (863, 908, 931, 832 g/d). ADFI decreased (linear, P = 0.014) with increasing LYS in control (2270, 2198, 2186, 2130 g/d), but increased (quadratic, P = 0.039) in DDGS diets (2112, 2207, 2324, 2103 g/d). DDGS reduced (P≤0.03) ADG (883 vs. 914 g/d), G:F (405 vs. 417 g/kg) and LEA (34.5 vs. 35.3 cm2), while increasing LYS decreased (linear, P < 0.005) G:F (417, 419, 409, 401 g/kg) and LEA (35.34, 35.17, 35.46, 33.64 cm2). Displacement of SBM with DDGS reduced growth and LYS addition negatively affected growth and G:F for diets with SBM, but not DDGS.

This study evaluated the effects of replacing soybean meal (SBM) with DDGS and crystalline amino acids on growth, carcass lean, and carcass yield of finishing pigs. Pigs (n = 480; 83.1±0.35 kg) were blocked by BW and sex and assigned to 80 pens (3 gilts and 3 barrows/pen). Treatments were arranged as a 2×4 factorial with DDGS included at 0 or 20% and L-lysine·HCl (LYS) added at 0.0, 0.2, 0.4, or 0.6%. Diets were balanced for ideal protein and NE and contained 0.75% and 0.67% SID lysine for Phase 1 (21 days) and Phase 2 (to market, 13 or 20 days), respectively. Only 0.49% LYS was necessary to meet the SID lysine requirement for the highest LYS (0.6%) diet for Phase 2. As LYS increased, dietary SBM inclusion decreased from 21.75% to 2.85% (Phase 1) and 18.75% to 3.35% (Phase 2) for control diets. It decreased from 18.40% to 0% (Phase 1) and 15.40 to 0% (Phase 2) for DDGS diets. During Phase 1, DDGS decreased ADG (992 vs. 1039 g/d; P = 0.031) and ADFI (3424 vs. 3503 g/d; P = 0.061). Increasing LYS linearly decreased ADG (1031, 1037, 1035, 959 g/d; P=0.026) and G:F (299, 295, 298, 281 g/kg; P = 0.026). Treatments did not impact Phase 2 performance. Overall, DDGS reduced ADG (1098 vs. 1131 g/d; P = 0.048) and ADFI (3638 vs 3712 g/d; P = 0.070). Supplemental LYS linearly decreased ADG (1133, 1141, 1120, and 1064 g/d; P = 0.005) and G:F (310, 305, 304, and 295 g/kg; P = 0.006). ADFI increased quadratically (P = 0.002) with LYS within SBM control, but not DDGS (interaction; P = 0.009). Ultrasound loin-eye-area corrected for BW tended to decrease quadratically (48.3, 48.8, 49.1, 47.4 cm2; P = 0.060) with increasing LYS. DDGS reduced carcass yield (72.62 vs. 73.04%; P = 0.034). Replacement of SBM with DDGS and high amino acid inclusion negatively impacted growth performance and carcass yield of finisher pigs.

Understanding how impacts may differ across alternative levels of future climate change is necessary to inform mitigation and adaptation measures. The Benefits of Reduced Anthropogenic Climate changE (BRACE) project assesses the differences in impacts between two specific climate futures: a higher emissions future with global average temperature increasing about 3.7 °C above pre-industrial levels toward the end of the century and a moderate emissions future with global average warming of about 2.5 °C. BRACE studies in this special issue quantify avoided impacts on physical, managed, and societal systems in terms of extreme events, health, agriculture, and tropical cyclones. Here we describe the conceptual framework and design of BRACE and synthesize its results. Methodologically, the project combines climate modeling, statistical analysis, and impact assessment and draws heavily on large ensembles using the Community Earth System Model. It addresses uncertainty in future societal change by employing two pathways for future socioeconomic development. Results show that the benefits of reduced climate change within this framework vary substantially across types of impacts. In many cases, especially related to extreme heat events, there are substantial benefits to mitigation. The benefits for some heat extremes are statistically significant in some regions as early as the 2020s and are widespread by mid-century. Benefits are more modest for agriculture and exposure to some health risks. Benefits are negative for agriculture when CO2 fertilization is incorporated. For several societal impacts, the effect on outcomes of alternative future societal development pathways is substantially larger than the effect of the two climate scenarios.

Unnatural base pairs (UBPs) have been developed and used for a variety of in vitro applications, as well as for the engineering of semi-synthetic organisms (SSOs) that store and retrieve increased information. However, these applications are limited by the availability of methods to rapidly and accurately determine the sequence of unnatural DNA. Here, we report the development and application of the MspA nanopore to sequence DNA containing the dTPT3-dNaM UBP. Analysis of two sequence contexts reveals that DNA containing the UBP is replicated with an efficiency and fidelity similar to that of natural DNA and sufficient for use as the basis of an SSO that produces proteins with non-canonical amino acids.

Outcomes after migraine surgery have been previously assessed using quantitative measurements, including the migraine headache index. Qualitative methodologies offer the ability to analyze patients' perceptions and pain experience, and may point to changes in domains not captured by quantitative instruments. The purpose of this study was to characterize individual patients' experiences with migraines and to analyze how patients' experience of headaches changes in relation to surgery.Patients who previously underwent migraine surgery performed by a single surgeon participated in semistructured interviews at least 1 year after surgery. Purposive sampling was used to recruit patients [n = 15 (73 percent female)]. Interviews were transcribed verbatim. A multidisciplinary team with backgrounds in surgery, pain management, medicine, and health services research coded and analyzed transcripts.Participants reported improvements in one or more domains of pain following surgery, and changes in medication use and effectiveness. Even in individuals with persistent pain postoperatively, surgery appeared to facilitate an improvement in headache self-efficacy, including an ability to participate in daily activities. Migraineurs frequently described a new degree of control over at least one aspect of their pain.Migraine surgery appears to positively impact patients' lives in ways that support and expand on previously published outcomes. Patients report benefiting from surgery in ways that are not currently captured in commonly used metrics. This study's findings support the need for more specific patient-reported outcome measures to help clinicians and patients understand the impact of surgery and which outcomes matter most to patients.

Understanding how rising temperatures, ocean acidification, and hypoxia affect the performance of coastal fishes is essential to predicting species-specific responses to climate change. Although a population&rsquo, s habitat influences physiological performance, little work has explicitly examined the multi-stressor responses of species from habitats differing in natural variability. Here, clearnose skate (Rostaraja eglanteria) and summer flounder (Paralichthys dentatus) from mid-Atlantic estuaries, and thorny skate (Amblyraja radiata) from the Gulf of Maine, were acutely exposed to current and projected temperatures (20, 24, or 28 &deg, C, 22 or 30 &deg, and 9, 13, or 15 &deg, C, respectively) and acidification conditions (pH 7.8 or 7.4). We tested metabolic rates and hypoxia tolerance using intermittent-flow respirometry. All three species exhibited increases in standard metabolic rate under an 8 &deg, C temperature increase (Q10 of 1.71, 1.07, and 2.56, respectively), although this was most pronounced in the thorny skate. At the lowest test temperature and under the low pH treatment, all three species exhibited significant increases in standard metabolic rate (44&ndash, 105%, p &lt, 0.05) and decreases in hypoxia tolerance (60&ndash, 84% increases in critical oxygen pressure, 0.05). This study demonstrates the interactive effects of increasing temperature and changing ocean carbonate chemistry are species-specific, the implications of which should be considered within the context of habitat.

Background: Peroneal pathology, including trauma, can result in Cavovarus-Drop Foot type deformity, precluding patients from functioning in or attempting bracing. Lateral ankle stabilization and other associated procedures sometimes are not enough to address the resultant deformity, thus many CavoVarus-Dropfoot patients still have gait abnormalities, function minimally in a brace and still have secondary ambulatory pain and loss of function even when braced. We review the results of 9 patients meeting this criteria and electing for a Modified Putti Procedure and lower extremity reconstruction. Materials and Methods: The charts of 9 patients with adult-acquired CavoVarus deformity foot type after peroneal rupture were reviewed, all but 1 had failed previous AFO/bracing. These patients underwent surgical correction all consisting of the Modified Putti procedure, along with other ancillary procedures and the results and outcomes were collected. Additionally, surgical technique was documented. Results: The mean VAS score improved from 9.1 to 2.4 pre- and post-operatively, respectively. The average time to weight bearing was 7.2 weeks. Four of 9 (44.44%) patients experienced some form of complication. Eight of 9 (88.88%) patients relayed the surgery was a success and would opt to undergo it again, given the choice. Conclusion: The Modified Putti Procedure showed promising results, great patient satisfaction and marked improvement in pre-operative and post-operative pain rating. This technique, utilized after severe peroneal trauma and resultant deformity is present, is a considerable option to have in the foot and ankle surgeon’s armament.

DEA tC is a tricyclic 2'-deoxycytidine analog that can be incorporated into oligonucleotides by solid-phase synthesis and that exhibits a large fluorescence enhancement when correctly base-paired with a guanine base in a DNA-DNA duplex. The synthesis of DEA tC begins with 5-amino-2-methylbenzothiazole and provides the DEA tC nucleobase analog over five synthetic steps. This nucleobase analog is then silylated using N,O-bis(trimethylsilyl)acetamide and conjugated to Hoffer's chlorosugar to provide the protected DEA tC nucleoside in good yield. Following protective-group removal and chromatographic isolation of the β-anomer, dimethoxytritylation and phosphoramidite synthesis offer the monomer for solid-phase DNA synthesis. Solid-phase DNA synthesis conditions using extended coupling of the DEA tC amidite and a short deprotection time are employed to maximize efficiency. By following the protocols described in this unit, the DEA tC fluorescent probe can be synthesized and can be incorporated into any desired synthetic DNA oligonucleotide. © 2018 by John Wiley & Sons, Inc.

Development of probes that allow for sequence-unrestricted recognition of double-stranded DNA (dsDNA) continues to attract much attention due to the prospect for molecular tools that enable detection, regulation, and manipulation of genes. We have recently introduced so-called Invader probes as alternatives to more established approaches such as triplex-forming oligonucleotides, peptide nucleic acids and polyamides. These short DNA duplexes are activated for dsDNA recognition by installment of +1 interstrand zippers of intercalator-functionalized nucleotides such as 2′-N-(pyren-1-yl)methyl-2′-N-methyl-2′-aminouridine and 2′-O-(pyren-1-yl)methyluridine, which results in violation of the nearest neighbor exclusion principle and duplex destabilization. The individual probes strands have high affinity toward complementary DNA strands, which generates the driving force for recognition of mixed-sequence dsDNA regions. In the present article, we characterize Invader probes that are based on phosphorothioate backbones (PS-DNA Invaders). The change from the regular phosphodiester backbone furnishes Invader probes that are much more stable to nucleolytic degradation, while displaying acceptable dsDNA-recognition efficiency. PS-DNA Invader probes therefore present themselves as interesting probes for dsDNA-targeting applications in cellular environments and living organisms.

Development of probes capable of recognizing specific regions of chromosomal DNA has been a long-standing goal for chemical biologists. Current strategies such as PNA, triplex-forming oligonucleotides, and polyamides are subject to target choice limitations and/or necessitate non-physiological conditions, leaving a need for alternative approaches. Toward this end, we have recently introduced double-stranded oligonucleotide probes that are energetically activated for DNA recognition through modification with +1 interstrand zippers of intercalator-functionalized nucleotide monomers. Herein, probes with different chemistries and architectures – varying in the position, number, and distance between the intercalator zippers – are studied with respect to hybridization energetics and DNA-targeting properties. Experiments with model DNA targets demonstrate that optimized probes enable efficient (C50 24 h), and single nucleotide specific recognition of DNA targets at physiologically relevant ionic strengths. Optimized probes were used in non-denaturing fluorescence in situ hybridization experiments for detection of gender-specific mixed-sequence chromosomal DNA target regions. These probes present themselves as a promising strategy for recognition of chromosomal DNA, which will enable development of new tools for applications in molecular biology, genomic engineering and nanotechnology.

Major efforts are currently being devoted to improving the binding affinity, target specificity, and enzymatic stability of oligonucleotides used for nucleic acid targeting applications in molecular biology, biotechnology, and medicinal chemistry. One of the most popular strategies toward this end has been to introduce additional modifications to the sugar ring of affinity-inducing conformationally restricted nucleotide building blocks such as locked nucleic acid (LNA). In the preceding article in this issue, we introduced a different strategy toward this end, i.e., C5-functionalization of LNA uridines. In the present article, we extend this strategy to α-L-LNA: i.e., one of the most interesting diastereomers of LNA. α-L-LNA uridine monomers that are conjugated to small C5-alkynyl substituents induce significant improvements in target affinity, binding specificity, and enzymatic stability relative to conventional α-L-LNA. The results from the back-to-back articles therefore suggest that C5-functionalization of pyrimidines is a general and synthetically straightforward approach to modulate biophysical properties of oligonucleotides modified with LNA or other conformationally restricted monomers.

Oligonucleotides modified with conformationally restricted nucleotides such as locked nucleic acid (LNA) monomers are used extensively in molecular biology and medicinal chemistry to modulate gene expression at the RNA level. Major efforts have been devoted to the design of LNA derivatives that induce even higher binding affinity and specificity, greater enzymatic stability, and more desirable pharmacokinetic profiles. Most of this work has focused on modifications of LNA's oxymethylene bridge. Here, we describe an alternative approach for modulation of the properties of LNA: i.e., through functionalization of LNA nucleobases. Twelve structurally diverse C5-functionalized LNA uridine (U) phosphoramidites were synthesized and incorporated into oligodeoxyribonucleotides (ONs), which were then characterized with respect to thermal denaturation, enzymatic stability, and fluorescence properties. ONs modified with monomers that are conjugated to small alkynes display significantly improved target affinity, binding specificity, and protection against 3'-exonucleases relative to regular LNA. In contrast, ONs modified with monomers that are conjugated to bulky hydrophobic alkynes display lower target affinity yet much greater 3'-exonuclease resistance. ONs modified with C5-fluorophore-functionalized LNA-U monomers enable fluorescent discrimination of targets with single nucleotide polymorphisms (SNPs). In concert, these properties render C5-functionalized LNA as a promising class of building blocks for RNA-targeting applications and nucleic acid diagnostics.

The development of molecular strategies that enable recognition of specific double-stranded DNA (dsDNA) regions has been a longstanding goal as evidenced by the emergence of triplex-forming oligonucleotides, peptide nucleic acids (PNAs), minor groove binding polyamides, and--more recently--engineered proteins such as CRISPR/Cas9. Despite this progress, an unmet need remains for simple hybridization-based probes that recognize specific mixed-sequence dsDNA regions under physiological conditions. Herein, we introduce pseudocomplementary Invader probes as a step in this direction. These double-stranded probes are chimeras between pseudocomplementary DNA (pcDNA) and Invader probes, which are activated for mixed-sequence dsDNA-recognition through the introduction of pseudocomplementary base pairs comprised of 2-thiothymine and 2,6-diaminopurine, and +1 interstrand zipper arrangements of intercalator-functionalized nucleotides, respectively. We demonstrate that certain pseudocomplementary Invader probe designs result in very efficient and specific recognition of model dsDNA targets in buffers of high ionic strength. These chimeric probes, therefore, present themselves as a promising strategy for mixed-sequence recognition of dsDNA targets for applications in molecular biology and nucleic acid diagnostics.

Targeting and invading double-stranded DNA with synthetic oligonucleotides under physiological conditions remain a challenge. Bis-locked nucleic acids (bisLNAs) are clamp-forming oligonucleotides able to invade into supercoiled DNA via combined Hoogsteen and Watson-Crick binding. To improve the bisLNA design, we investigated its mechanism of binding. Our results suggest that bisLNAs bind via Hoogsteen-arm first, followed by Watson-Crick arm invasion, initiated at the tail. Based on this proposed hybridization mechanism, we designed next-generation bisLNAs with a novel linker able to stack to adjacent nucleobases, a new strategy previously not applied for any type of clamp-constructs. Although the Hoogsteen-arm limits the invasion, upon incorporation of the stacking linker, bisLNA invasion is significantly more efficient than for non-clamp, or nucleotide-linker containing LNA-constructs. Further improvements were obtained by substituting LNA with 2'-glycylamino-LNA, contributing a positive charge. For regular bisLNAs a 14-nt tail significantly enhances invasion. However, when two stacking linkers were incorporated, tail-less bisLNAs were able to efficiently invade. Finally, successful targeting of plasmids inside bacteria clearly demonstrates that strand invasion can take place in a biologically relevant context.

Toxoplasma gondii is an obligate intracellular protozoan parasite whose rapid lytic replication cycles define its pathogenicity. We identified a temperature-sensitive growth mutant, FV-P6, which irreversibly arrests before the middle of the G1 stage of the tachyzoite cell cycle. This arrest is caused by a point mutation in a gene conserved across eukaryotes, Cactin, whose product localizes to the nucleus. To elucidate the role of TgCactin we performed genome-wide expression profiling. Besides the expected G1 expression profile, many genes associated with the extracellular state as well as with the bradyzoite cyst stage were identified. Consistent with these profiles were the expression of AP2 transcription factors typically associated with extracellular and bradyzoite stage parasites. This suggests a role for TgCactin in control of gene expression. As TgCactin does not contain any functionally defined domains we reasoned TgCactin exerts its function through interactions with other proteins. In support of this model we demonstrated that TgCactin is present in a protein complex and can oligomerize. Taken together, these results suggest that TgCactin acts as a pivotal protein potentially regulating gene expression at several transition points in parasite development.

The intracellular protozoan parasite Toxoplasma gondii divides by a unique process of internal budding that involves the assembly of two daughter cells within the mother. The cytoskeleton of Toxoplasma, which is composed of microtubules associated with an inner membrane complex (IMC), has an important role in this process. The IMC, which is directly under the plasma membrane, contains a set of flattened membranous sacs lined on the cytoplasmic side by a network of filamentous proteins. This network contains a family of intermediate filament-like proteins or IMC proteins. In order to elucidate the division process, we have characterized a 14-member subfamily of Toxoplasma IMC proteins that share a repeat motif found in proteins associated with the cortical alveoli in all alveolates. By creating fluorescent protein fusion reporters for the family members we determined the spatiotemporal patterns of all 14 IMC proteins through tachyzoite development. This revealed several distinct distribution patterns and some provide the basis for novel structural models such as the assembly of certain family members into the basal complex. Furthermore we identified IMC15 as an early marker of budding and, lastly, the dynamic patterns observed throughout cytokinesis provide a timeline for daughter parasite development and division.

Udgivelsesdato: Nov-23

Pyrene-functionalized oligonucleotides are intensively explored for applications in materials science and diagnostics. Here, we describe a short synthetic route to 2′-S-(pyren-1-yl)methyl-2′-thiouridine monomer S, its incorporation into oligodeoxyribonucleotides (ONs), and biophysical characterization thereof. Pseudorotational analysis reveals that the furanose ring of this monomer has a slight preference for South-type conformations. ONs modified with monomer S display high cDNA affinity but decreased binding specificity. Hybridization is associated with bathochromic shifts of pyrene absorption bands and quenching of pyrene fluorescence consistent with an intercalative binding mode of the pyrene moiety. Monomer S was also evaluated as a building block for mixed-sequence recognition of double-stranded DNA via the Invader strategy. However, probes with +1 interstrand arrangements of monomer S were found to be less efficient than Invader probes based on 2′-O-(pyren-1-yl)methyluridine or 2′-N-(pyren-1-yl)methyl-2′-N-methyl-2′-aminouridine.

Invader probes have been proposed as alternatives to polyamides, triplex-forming oligonucleotides, and peptide nucleic acids for recognition of chromosomal DNA targets. These double-stranded probes are activated for DNA recognition by +1 interstrand zippers of pyrene-functionalized nucleotides. This particular motif forces the intercalating pyrene moieties into the same region, resulting in perturbation and destabilization of the probe duplex. In contrast, the two probe strands display very high affinity toward complementary DNA. The energy difference between the probe duplexes and recognition complexes provides the driving force for DNA recognition. In the present study, we explore the properties of Invader probes based on larger intercalators, i.e., perylene and coronene, expecting that the larger π-surface area will result in additional destabilization of the probe duplex and further stabilization of probe-target duplexes, in effect increasing the thermodynamic driving force for DNA recognition. Toward this end, we developed protocols for 2'-N-methyl-2'-amino-2'-deoxyuridine phosphoramidites that are functionalized at the N2'-position with pyrene, perylene, or coronene moieties and incorporated these monomers into oligodeoxyribonucleotides (ONs). The resulting ONs and Invader probes are characterized by thermal denaturation experiments, analysis of thermodynamic parameters, absorption and fluorescence spectroscopy, and DNA recognition experiments. Invader probes based on large intercalators efficiently recognize model targets.

The ISS and the prior station Mir provided the proving ground for future human long-duration space activity. A recent European Space Agency study recommended “Measurement campaigns on the ISS form the ideal tool for experimental validation of radiation environment models, of transport code algorithms and reaction cross sections”. Indeed, prior measurements on Shuttle have provided vital information impacting both transport code and environmental model development. Recent studies using the ISS 7A configuration with TLD area monitors demonstrated that computational dosimetry requires environmental models with accurate anisotropic and dynamic behavior, detailed information on rack loading, and an accurate 6 degree-of-freedom description of the ISS trajectory. The ISS model is now configured for 11A and uses an anisotropic and dynamic geomagnetic transmission and trapped proton models. The ISS 11A is instrumented with both passive and active dosimetric devices. Time resolved measurements have the advantage of isolating trapped proton and galactic cosmic ray components as was essential to transport code validation in Shuttle data analysis. ISS 11A model validation will begin with passive dosimetry as was used with ISS 7A. Directional dependent active measurements will play an important role in the validation of environmental model anisotropies.

Ongoing assembly and maintenance activities at the International Space Station (ISS) require much more extravehicular activity (EVA) than did the earlier U.S. Space Shuttle missions. It is thus desirable to determine and analyze, and possibly foresee, as accurately as possible what radiation exposures crew members involved in EVAs will experience in order to minimize risks and to establish exposure limits that must not to be exceeded. A detailed CAD model of the U.S. Space Shuttle EVA Spacesuit, developed at NASA Langley Research Center (LaRC), is used to represent the directional shielding of an astronaut; it has detailed helmet and backpack structures, hard upper torso, and multilayer space suit fabric material. The NASA Computerized Anatomical Male and Female (CAM and CAF) models are used in conjunction with the space suit CAD model for dose evaluation within the human body. The particle environments are taken from the orbit-averaged NASA AP8 and AE8 models at solar cycle maxima and minima. The transport of energetic particles through space suit materials and body tissue is calculated by using the NASA LaRC HZETRN code for hadrons and a recently developed deterministic transport code, ELTRN, for electrons. The doses within the CAM and CAF models are determined from energy deposition at given target points along 968 directional rays convergent on the points and are evaluated for several points on the skin and within the body. Dosimetric quantities include contributions from primary protons, light ions, and electrons, as well as from secondary brehmsstrahlung and target fragments. Directional dose patterns are displayed as rays and on spherical surfaces by the use of a color relative intensity representation.

Oligonucleotides modified with C5-amino acid LNA uridine result in highly thermostable duplexes, with excellent discrimination of single nucleotide mismatches. Additionally, these ONs are inert toward 3′-exonuclease degradation.

The development of conformationally restricted nucleotide building blocks continues to attract considerable interest because of their successful use within antisense, antigene, and other gene-targeting strategies. Locked nucleic acid (LNA) and its diastereomer α-l-LNA are two interesting examples thereof. Oligonucleotides modified with these units display greatly increased affinity toward nucleic acid targets, improved binding specificity, and enhanced enzymatic stability relative to unmodified strands. Here we present the synthesis and biophysical characterization of oligodeoxyribonucleotides (ONs) modified with 2′-amino-α-l-LNA adenine monomers W–Z. The synthesis of the target phosphoramidites 1–4 is initiated from pentafuranose 5, which upon Vorbruggen glycosylation, O2′-deacylation, O2′-activation and C2′-azide introduction yields nucleoside 8. A one-pot tandem Staudinger/intramolecular nucleophilic substitution converts 8 into 2′-amino-α-l-LNA adenine intermediate 9, which after a series of nontrivia...

A short and high-yielding synthetic route to [3-15N]-labeled uridine phosphoramidite 1 (26% overall yield from uridine) has been developed. This will enable automated synthesis of isotopically labeled RNA strands and facilitate their use in structural studies.

The development of synthetic agents that recognize double-stranded DNA (dsDNA) is a long-standing goal that is inspired by the promise for tools that detect, regulate, and modify genes. Progress has been made with triplex-forming oligonucleotides, peptide nucleic acids, and polyamides, but substantial efforts are currently devoted to the development of alternative strategies that overcome the limitations observed with the classic approaches. In 2005, we introduced Invader locked nucleic acids (LNAs), i.e., double-stranded probes that are activated for mixed-sequence recognition of dsDNA through modification with "+1 interstrand zippers" of 2'-N-(pyren-1-yl)methyl-2'-amino-α-l-LNA monomers. Despite promising preliminary results, progress has been slow because of the synthetic complexity of the building blocks. Here we describe a study that led to the identification of two simpler classes of Invader monomers. We compare the thermal denaturation characteristics of double-stranded probes featuring different interstrand zippers of pyrene-functionalized monomers based on 2'-amino-α-l-LNA, 2'-N-methyl-2'-amino-DNA, and RNA scaffolds. Insights from fluorescence spectroscopy, molecular modeling, and NMR spectroscopy are used to elucidate the structural factors that govern probe activation. We demonstrate that probes with +1 zippers of 2'-O-(pyren-1-yl)methyl-RNA or 2'-N-methyl-2'-N-(pyren-1-yl)methyl-2'-amino-DNA monomers recognize DNA hairpins with similar efficiency as original Invader LNAs. Access to synthetically simple monomers will accelerate the use of Invader-mediated dsDNA recognition for applications in molecular biology and nucleic acid diagnostics.

The development of conformationally restricted nucleotide building blocks continues to attract considerable interest because of their successful use within antisense, antigene, and other gene-targeting strategies. Locked nucleic acid (LNA) and its diastereomer α-L-LNA are two interesting examples thereof. Oligonucleotides modified with these units display greatly increased affinity toward nucleic acid targets, improved binding specificity, and enhanced enzymatic stability relative to unmodified strands. Here we present the synthesis and biophysical characterization of oligodeoxyribonucleotides (ONs) modified with 2'-amino-α-L-LNA adenine monomers W-Z. The synthesis of the target phosphoramidites 1-4 is initiated from pentafuranose 5, which upon Vorbrüggen glycosylation, O2'-deacylation, O2'-activation and C2'-azide introduction yields nucleoside 8. A one-pot tandem Staudinger/intramolecular nucleophilic substitution converts 8 into 2'-amino-α-L-LNA adenine intermediate 9, which after a series of nontrivial protecting-group manipulations affords key intermediate 15. Subsequent chemoselective N2'-functionalization and O3'-phosphitylation give targets 1-4 in ~1-3% overall yield over 11 steps from 5. ONs modified with pyrene-functionalized 2'-amino-α-L-LNA adenine monomers X-Z display greatly increased affinity toward DNA targets (ΔTm/modification up to +14 °C). Results from absorption and fluorescence spectroscopy suggest that the duplex stabilization is a result of pyrene intercalation. These characteristics render N2'-pyrene-functionalized 2'-amino-α-L-LNAs of considerable interest for DNA-targeting applications.