Modifying the specificity of an RNA backbone contact 1 1Edited by D. E. Draper

Dertinger, Dagmar; Dale, Taraka; Uhlenbeck, Olke C.

doi:10.1006/jmbi.2001.5132

Cited by 15 publications

(15 citation statements)

References 18 publications

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“…A comparable result was obtained for the GluA63Gln, GluA63Ala and GluA63Asp mutant proteins, where substitution of GluA63 disrupted the only contact of the MS2 coat protein with a deoxyribose group of the RNA operator, but left other areas of the RNA-protein complex unchanged, as confirmed by the binding affinities of a panel of phosphorothioate containing RNAs. 53 In contrast, the remaining three mutant proteins examined, AsnB55Ala, LysA61Ala, and LysA57Ala, showed a quite different set of R values than the wild-type protein, suggesting that they interact with an altered thermodynamic profile (Figure 3(d)-(f)). While the AsnB55Ala protein showed a modified R value for one of the phosphorothioate isomers at position K7, where the RNA contact with AsnB55 is missing, it also showed modified R values at two other sites, the O1 of phosphate K5 and the 2 0 -hydroxyl of base K5.…”

Section: Combining Mutant Proteins With Modified Rnasmentioning

confidence: 81%

“…10,11,53 Hairpin 1 is a 15 nucleotide RNA that contains all the sequence elements required for recognition by the protein, while hairpin 2 ( Figure 3) has an additional cytidine at the 5 0 -end, which allows for the introduction of a phosphorothioate at position K13. 10 All RNAs used in this study were labeled at their 5 0 -end with T4 polynucleotide kinase and [g-32 P]ATP 60 and subsequently purified by electrophoresis on a denaturing 20% (w/v) polyacrylamide gel.…”

Section: Methodsmentioning

confidence: 99%

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Alanine Scanning of MS2 Coat Protein Reveals Protein–Phosphate Contacts Involved in Thermodynamic Hot Spots

Hobson

Uhlenbeck

2006

Journal of Molecular Biology

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Section: Combining Mutant Proteins With Modified Rnasmentioning

confidence: 81%

Section: Methodsmentioning

confidence: 99%

Alanine Scanning of MS2 Coat Protein Reveals Protein–Phosphate Contacts Involved in Thermodynamic Hot Spots

Hobson

Uhlenbeck

2006

Journal of Molecular Biology

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“…Modifications of RNA at the 2'-position of the ribose ring increase siRNA stability by denying the action site to endonucleases and reducing immune response activation. The resulting molecule is resistant to nucleases and has an increased half-life in vivo (141,142). Apart from modifying the chemical bond, the sugar unit may also be modified as in 2′-fluoro (2′-F), 2′-O-fluoro-β-D-arabinonucleotide (FANA) and 2′-O-(2-methoxyethyl) (MOE) as well as during synthesis of modified RNA nucleotides termed locked nucleic acid (LNA).…”

Section: Backbone and Structural Modificationmentioning

confidence: 99%

Subcellular Fate and Off-Target Effects of siRNA, shRNA, and miRNA

2011

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RNA interference (RNAi) strategies include double-stranded RNA (dsRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), and microRNA (miRNA). As this is a highly specific technique, efforts have been made to utilize RNAi towards potential knock down of disease-causing genes in a targeted fashion. RNAi has the potential to selectively inhibit gene expression by degrading or blocking the translation of the target mRNA. However, delivering these RNAs to specific cells presents a significant challenge. Some of these challenges result from the necessity of traversing the circulatory system while avoiding kidney filtration, degradation by endonucleases, aggregation with serum proteins, and uptake by phagocytes. Further, non-specific delivery may result in side-effects, including the activation of immune response. We discuss the challenges in the systemic delivery to target cells, cellular uptake, endosomal release and intracellular transport of RNAi drugs and recent progress in overcoming these barriers. We also discuss approaches that increase the specificity and metabolic stability and reduce the off-target effects of RNAi strategy.

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“…Specific and nonspecific capsid protein-oligonucleotide interactions were also analyzed in detail in artificial complexes formed by a recombinant capsid of the bacteriophage MS2 and identical copies of a unique viral ssRNA fragment (OR) involved in translational repression and initiation of capsid assembly (31,32). This allowed a structural interpretation of mutational analyses on the formation of a biologically relevant complex between OR and an MS2 capsid protein dimer (32)(33)(34)(35)(36)(37).…”

mentioning

confidence: 99%

Functional Relevance of Amino Acid Residues Involved in Interactions with Ordered Nucleic Acid in a Spherical Virus

Reguera

Grueso

Carreira

et al. 2005

Journal of Biological Chemistry

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In the spherical virion of the parvovirus minute virus of mice, several amino acid side chains of the capsid were previously found to be involved in interactions with the viral single-stranded DNA molecule. We have individually truncated by mutation to alanine many (ten) of these side chains and analyzed the effects on capsid assembly, stability and conformation, viral DNA encapsidation, and virion infectivity. Mutation of residues Tyr-270, Asp-273, or Asp-474 led to a drastic reduction in infectivity. Mutant Y270A was defective in capsid assembly; mutant D273A formed stable capsids, but it was essentially unable to encapsidate the viral DNA or to externalize the N terminus of the capsid protein VP2, a connected conformational event. Mutation of residues Asp-58, Trp-60, Asn-183, Thr-267, or Lys-471 led to a moderate reduction in infectivity. None of these mutations had an effect on capsid assembly or stability, or on the DNA encapsidation process. However, those five mutant virions were substantially less stable than the parental virion in thermal inactivation assays. The results with this model spherical virus indicate that several capsid residues that are found to be involved in polar interactions or multiple hydrophobic contacts with the viral DNA molecule contribute to preserving the active conformation of the infectious viral particle. Their effect appears to be mediated by the non-covalent interactions they establish with the viral DNA. In addition, at least one acidic residue at each DNA-binding region is needed for DNA packaging.Many fundamental biological processes involve protein-nucleic acid recognition. Double-stranded DNA-protein interactions have been extensively studied, but single-stranded (ss) 1 DNA-or RNA-protein interactions are much less characterized. In particular, very little is known about the molecular interactions between the nucleic acid genome of ssDNA or RNA virus and its protein shell, or the functional role of those interactions.Such knowledge may be essential for a better understanding of the life cycle of viruses, and for the design of drugs aimed at disrupting nucleic acid-protein interfaces.The structure of a helical virus, tobacco mosaic virus, has revealed defined interactions between repeating sets of three nucleotides in its ssRNA genome and each capsid subunit (reviewed in Ref. 1). In the crystal structures of spherical viruses (reviewed in Refs. 2-6) a large part, or all, of the nucleic acid component is invisible, because it is randomly oriented within the viral particles that form the crystal. Fortunately, in a few icosahedral viruses some segments of the nucleic acid molecule (ϳ10 -60% of the total) are arranged with the same symmetry as the capsid, and could be visualized. The structural analysis (reviewed in Refs. 4, 7-10) has revealed that the visible capsidnucleic acid interfaces generally include a limited number of amino acids and some 7-30 nucleotides that in several, but not in all cases, form intramolecular double-helical segments. The tertiary structure acqui...

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Modifying the specificity of an RNA backbone contact 1 1Edited by D. E. Draper

Cited by 15 publications

References 18 publications

Alanine Scanning of MS2 Coat Protein Reveals Protein–Phosphate Contacts Involved in Thermodynamic Hot Spots

Alanine Scanning of MS2 Coat Protein Reveals Protein–Phosphate Contacts Involved in Thermodynamic Hot Spots

Subcellular Fate and Off-Target Effects of siRNA, shRNA, and miRNA

Functional Relevance of Amino Acid Residues Involved in Interactions with Ordered Nucleic Acid in a Spherical Virus

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