85 research outputs found
Influence of a fluorobenzene nucleobase analogue on the conformational flexibility of RNA studied by molecular dynamics simulations
Chemically modified bases are frequently used to stabilize nucleic acids, to study the driving forces for nucleic acid structure formation and to tune DNA and RNA hybridization conditions. In particular, fluorobenzene and fluorobenzimidazole base analogues can act as universal bases able to pair with any natural base and to stabilize RNA duplex formation. Although these base analogues are compatible with an A-form RNA geometry, little is known about the influence on the fine structure and conformational dynamics of RNA. In the present study, nano-second molecular dynamics (MD) simulations have been performed to characterize the dynamics of RNA duplexes containing a central 1'-deoxy-1'-(2,4-difluorophenyl)-Ć-D-ribofuranose base pair or opposite to an adenine base. For comparison, RNA with a central uridine:adenine pair and a 1'-deoxy-1'-(phenyl)-Ć-D-ribofuranose opposite to an adenine was also investigated. The MD simulations indicate a stable overall A-form geometry for the RNAs with base analogues. However, the presence of the base analogues caused a locally enhanced mobility of the central bases inducing mainly base pair shear and opening motions. No stable ābase-pairedā geometry was found for the base analogue pair or the base analogue:adenine pairs, which explains in part the universal base character of these analogues. Instead, the conformational fluctuations of the base analogues lead to an enhanced accessibility of the bases in the major and minor grooves of the helix compared with a regular base pair
Synthesis of 1,4-Disubstituted Mono and Bis-triazolocarbo-acyclonucleoside Analogues of 9-(4-Hydroxybutyl)guanine by Cu(I)-Catalyzed Click Azide-Alkyne Cycloaddition
A series of novel mono-1,2,3-triazole and bis-1,2,3-triazole acyclonucleoside analogues of 9-(4-hydroxybutyl)guanine was prepared via copper(I)-catalyzed 1,3-dipolar cycloaddition of N-9 propargylpurine, N-1-propargylpyrimidines/as-triazine with the azido-pseudo-sugar 4-azidobutylacetate under solvent-free microwave conditions, followed by treatment with K2CO3/MeOH, or NH3/MeOH. All compounds studied in this work were screened for their antiviral activities [against human rhinovirus (HRV) and hepatitis C virus (HCV)] and antibacterial activities against a series of Gram positive and negative bacteria
Meeting review : ESF workshop on "Impact of nucleic acid chemistry on gene function analysis: antisense, aptamers, ribozymes and RNAi"
The shortage of functional information compared to the abundance of sequence information characterizes todayās situation in functional genomics. For many years the knock-down of a geneās product has been the most powerful way of analysing its function. In addition to the complete knock-out by homologous recombination, several different techniques have been developed to temporarily knock down gene expression through methods based on specific sequence recognition, such as knockdown by antisense oligonucleotides, ribozymes, aptamers or RNAi.
The ESF workshop on āImpact of Nucleic Acid Chemistry on Gene Function Analysisā brought together researchers who use techniques that are different but highly related. It offered an opportunity for an in-depth discussion of recent progress and common problems. Antisense oligonucleotides aptamers and ribozymes are techniques that have been used successfully for many years to validate targets. However, recent developments, such as increased tightness of binding (e.g. locked nucleic acids) or the combination of different methods (e.g. using aptamers to design ribozymes), have continued to improve the existing techniques. RNA interference (RNAi) is a defence mechanism of the cell against viruses. Since the exact mechanism of action within the cell is still unclear, RNAi was a particularly exciting topic at the workshop and was addressed in the largest number of presentations. Predictability of positional effects (accessibility of RNA) is a problem shared by all techniques using sequence-specific recognition and was the subject of quite controversial debates.
The meeting comprised over 50 people from 14 countries (13 European countries and the USA)
3-Ethynyl-2,2,5,5-tetraĀmethyl-1-oxyl-3-pyrroline
The five-membered ring of the title compound, C10H14NO, is almost planar [mean deviation from best plane = 0.006ā
(1)ā
Ć
]. The NāO bond is in the plane of the five-membered ring. The molĀecule is positioned about a pseudo-mirror plane at y = 0.375. In the crystal, molĀecules are connected by interĀmolecular CāHāÆO contacts into layers parallel to (010)
2-({4-[4-(1H-Benzimidazol-2-yl)phenĀyl]-1H-1,2,3-triazol-1-yl}methĀoxy)ethanol
In the title molecule, C18H17N5O2, the dihedral angle between the benzene plane and the benzimidazole plane is 19.8ā
(1)Ā° and the angle between the benzene plane and the triazole plane is 16.7ā
(1)Ā°. In the crystal, molĀecules are connected by OāHāÆN hydrogen bonds, forming zigzag chains along the c-axis direction. The chains are connected by bifurcated NāHāÆ(N,N) hydrogen bonds into layers parallel to (100). These layers are connected along the a-axis direction by weak CāHāÆO contacts, forming a three-dimensional network
4-Ethynyl-2,2,6,6-tetraĀmethyl-1,2,5,6-tetraĀhydroĀpyridine N-oxide
The six-membered ring of the title compound, C11H16NO, has a distorted envelope conformation. The piperidine N atom deviates by 0.128ā
(1)ā
Ć
from the plane through its three neighbouring atoms. In the crystal structure, molĀecules are connected by interĀmolecular CethynĀylāHāÆO contacts to form chains extending in the [10] direction
Exploration of factors driving incorporation of unnatural dNTPS into DNA by Klenow fragment (DNA polymerase I) and DNA polymerase Ī±
In order to further understand how DNA polymerases discriminate against incorrect dNTPs, we synthesized two sets of dNTP analogues and tested them as substrates for DNA polymerase Ī± (pol Ī±) and Klenow fragment (exo(ā)) of DNA polymerase I (Escherichia coli). One set of analogues was designed to test the importance of the electronic nature of the base. The bases consisted of a benzimidazole ring with one or two exocyclic substituent(s) that are either electron-donating (methyl and methoxy) or electron-withdrawing (trifluoromethyl and dinitro). Both pol Ī± and Klenow fragment exhibit a remarkable inability to discriminate against these analogues as compared to their ability to discriminate against incorrect natural dNTPs. Neither polymerase shows any distinct electronic or steric preferences for analogue incorporation. The other set of analogues, designed to examine the importance of hydrophobicity in dNTP incorporation, consists of a set of four regioisomers of trifluoromethyl benzimidazole. Whereas pol Ī± and Klenow fragment exhibited minimal discrimination against the 5- and 6-regioisomers, they discriminated much more effectively against the 4- and 7-regioisomers. Since all four of these analogues will have similar hydrophobicity and stacking ability, these data indicate that hydrophobicity and stacking ability alone cannot account for the inability of pol Ī± and Klenow fragment to discriminate against unnatural bases. After incorporation, however, both sets of analogues were not efficiently elongated. These results suggest that factors other than hydrophobicity, sterics and electronics govern the incorporation of dNTPs into DNA by pol Ī± and Klenow fragment
The ribosome assembly factor Nep1 responsible for BowenāConradi syndrome is a pseudouridine-N1-specific methyltransferase
Nep1 (Emg1) is a highly conserved nucleolar protein with an essential function in ribosome biogenesis. A mutation in the human Nep1 homolog causes BowenāConradi syndromeāa severe developmental disorder. Structures of Nep1 revealed a dimer with a fold similar to the SPOUT-class of RNA-methyltransferases suggesting that Nep1 acts as a methyltransferase in ribosome biogenesis. The target for this putative methyltransferase activity has not been identified yet. We characterized the RNA-binding specificity of Methanocaldococcus jannaschii Nep1 by fluorescence- and NMR-spectroscopy as well as by yeast three-hybrid screening. Nep1 binds with high affinity to short RNA oligonucleotides corresponding to nt 910ā921 of M. jannaschii 16S rRNA through a highly conserved basic surface cleft along the dimer interface. Nep1 only methylates RNAs containing a pseudouridine at a position corresponding to a previously identified hypermodified N1-methyl-N3-(3-amino-3-carboxypropyl) pseudouridine (m1acp3-ĪØ) in eukaryotic 18S rRNAs. Analysis of the methylated nucleoside by MALDI-mass spectrometry, HPLC and NMR shows that the methyl group is transferred to the N1 of the pseudouridine. Thus, Nep1 is the first identified example of an N1-specific pseudouridine methyltransferase. This enzymatic activity is also conserved in human Nep1 suggesting that Nep1 is the methyltransferase in the biosynthesis of m1acp3-ĪØ in eukaryotic 18S rRNAs
A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects
Small interfering RNAs (siRNAs) are now established as the preferred tool to inhibit gene function in mammalian cells yet trigger unintended gene silencing due to their inherent miRNA-like behavior. Such off-target effects are primarily mediated by the sequence-specific interaction between the siRNA seed regions (position 2ā8 of either siRNA strand counting from the 5ā²-end) and complementary sequences in the 3ā²UTR of (off-) targets. It was previously shown that chemical modification of siRNAs can reduce off-targeting but only very few modifications have been tested leaving more to be identified. Here we developed a luciferase reporter-based assay suitable to monitor siRNA off-targeting in a high throughput manner using stable cell lines. We investigated the impact of chemically modifying single nucleotide positions within the siRNA seed on siRNA function and off-targeting using 10 different types of chemical modifications, three different target sequences and three siRNA concentrations. We found several differently modified siRNAs to exercise reduced off-targeting yet incorporation of the strongly destabilizing unlocked nucleic acid (UNA) modification into position 7 of the siRNA most potently reduced off-targeting for all tested sequences. Notably, such position-specific destabilization of siRNAātarget interactions did not significantly reduce siRNA potency and is therefore well suited for future siRNA designs especially for applications in vivo where siRNA concentrations, expectedly, will be low
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