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Synthesis of native-like crosslinked duplex RNA and study of its properties.

ABSTRACT: A variety of enzymes have been found to interact with double-stranded RNA (dsRNA) in order to carry out its functions. We have endeavored to prepare the covalently crosslinked native-like duplex RNA, which could be useful for biochemical studies and RNA nanotechnology. In this study, the interstrand covalently linked duplex RNA was formed by a crosslinking reaction between vinylpurine (VP) and the target cytosine or uracil in RNA. We measured melting temperatures and CD spectra to identify the properties of the VP crosslinked duplex RNA. The crosslinking formation increased the thermodynamic stability without disturbing the natural conformation of dsRNA. In addition, a competitive binding experiment with the duplex RNA binding enzyme, ADAR2, showed the crosslinked dsRNA bound the protein with nearly the same binding affinity as the natural dsRNA, confirming that it has finely preserved the natural traits of duplex RNA.

SUBMITTER: Onizuka K

PROVIDER: S-EPMC5969911 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Synthesis of native-like crosslinked duplex RNA and study of its properties.

Onizuka Kazumitsu K Hazemi Madoka E ME Thomas Justin M JM Monteleone Leanna R LR Yamada Ken K Imoto Shuhei S Beal Peter A PA Nagatsugi Fumi F

Bioorganic & medicinal chemistry 20170221 7

A variety of enzymes have been found to interact with double-stranded RNA (dsRNA) in order to carry out its functions. We have endeavored to prepare the covalently crosslinked native-like duplex RNA, which could be useful for biochemical studies and RNA nanotechnology. In this study, the interstrand covalently linked duplex RNA was formed by a crosslinking reaction between vinylpurine (VP) and the target cytosine or uracil in RNA. We measured melting temperatures and CD spectra to identify the p ...[more]

PMID: 28268052

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Project description:Alginate hydrogels have been investigated for a broad variety of medical applications. The ability to assemble hydrogels at neutral pH and mild temperatures makes alginate a popular choice for the encapsulation and delivery of cells and proteins. Alginate has been studied extensively for the delivery of islets as a treatment for type 1 diabetes. However, poor stability of the encapsulation systems after implantation remains a challenge. In this paper, alginate was modified with 2-aminoethyl methacrylate hydrochloride (AEMA) to introduce groups that can be photoactivated to generate covalent bonds. This enabled formation of dual crosslinked structure upon exposure to ultraviolet light following initial ionic crosslinking into bead structures. The degree of methacrylation was varied and in vitro stability, long term swelling, and cell viability examined. At low levels of the methacrylation, the beads could be formed by first ionic crosslinks followed by exposure to ultraviolet light to generate covalent bonds. The methacrylated alginate resulted in more stable beads and cells were viable following encapsulation. Alginate microbeads, ionic (unmodified) and dual crosslinked, were implanted into a rat omentum pouch model. Implantation was performed with a local injection of 100?µl of 50?µg/ml of Lipopolysaccharide (LPS) to stimulate a robust inflammatory challenge in vivo. Implants were retrieved at 1 and 3?weeks for analysis. The unmodified alginate microbeads had all failed by week 1, whereas the dual-crosslinked alginate microbeads remained stable up through 3?weeks. The modified alginate microbeads may provide a more stable alternative to current alginate-based systems for cell encapsulation. STATEMENT OF SIGNIFICANCE:Alginate, a naturally occurring polysaccharide, has been used for cell encapsulation to prevent graft rejection of cell transplants for people with type I diabetes. Although some success has been observed in clinical trials, the lack of reproducibility and failure to reach insulin dependence for longer periods of time indicates the need for improvements in the procedure. A major requirement for the long-term function of alginate encapsulated cells is the mechanical stability of microcapsules. Insufficient mechanical integrity of the capsules can lead to immunological reactions in the recipients. In this work, alginate was modified to allow photoactivatable groups in order to allow formation of covalent crosslinks in addition to ionic crosslinking. The dual crosslinking design prevents capsule breakdown following implantation in vivo.

Project description:It is generally thought that the promoters of non-segmented, negative strand RNA viruses (nsNSVs) direct the polymerase to initiate RNA synthesis exclusively opposite the 3´ terminal nucleotide of the genome RNA by a de novo (primer independent) initiation mechanism. However, recent studies have revealed that there is diversity between different nsNSVs with pneumovirus promoters directing the polymerase to initiate at positions 1 and 3 of the genome, and ebolavirus polymerases being able to initiate at position 2 on the template. Studies with other RNA viruses have shown that polymerases that engage in de novo initiation opposite position 1 typically have structural features to stabilize the initiation complex and ensure efficient and accurate initiation. This raised the question of whether different nsNSV polymerases have evolved fundamentally different structural properties to facilitate initiation at different sites on their promoters. Here we examined the functional properties of polymerases of respiratory syncytial virus (RSV), a pneumovirus, human parainfluenza virus type 3 (PIV-3), a paramyxovirus, and Marburg virus (MARV), a filovirus, both on their cognate promoters and on promoters of other viruses. We found that in contrast to the RSV polymerase, which initiated at positions 1 and 3 of its promoter, the PIV-3 and MARV polymerases initiated exclusively at position 1 on their cognate promoters. However, all three polymerases could recognize and initiate from heterologous promoters, with the promoter sequence playing a key role in determining initiation site selection. In addition to examining de novo initiation, we also compared the ability of the RSV and PIV-3 polymerases to engage in back-priming, an activity in which the promoter template is folded into a secondary structure and nucleotides are added to the template 3´ end. This analysis showed that whereas the RSV polymerase was promiscuous in back-priming activity, the PIV-3 polymerase generated barely detectable levels of back-primed product, irrespective of promoter template sequence. Overall, this study shows that the polymerases from these three nsNSV families are fundamentally similar in their initiation properties, but have differences in their abilities to engage in back-priming.

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Synthesis of native-like crosslinked duplex RNA and study of its properties.

Publications

Synthesis of native-like crosslinked duplex RNA and study of its properties.

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