Biomedical subjects

Charles R Allerson

Publications and source records attributed to Charles R Allerson.

6 recordsLinked to original sources

PubMed2006

Competition for RISC binding predicts in vitro potency of siRNA.

Short interfering RNAs (siRNA) guide degradation of target RNA by the RNA-induced silencing complex (RISC). The use of siRNA in animals is limited partially due to the short half-life of siRNAs in tissues. Chemically modified siRNAs are necessary that maintain mRNA degradation activity, but are more stable to nucleases. In this study, we utilized alternating 2'-O-methyl and 2'-deoxy-2'-fluoro (OMe/F) chemically modified siRNA targeting PTEN and Eg5. OMe/F-modified siRNA consistently reduced mRNA and protein levels with equal or greater potency and efficacy than unmodified siRNA. We showed that modified siRNAs use the RISC mechanism and lead to cleavage of target mRNA at the same position as unmodified siRNA. We further demonstrated that siRNAs can compete with each other, where highly potent siRNAs can compete with less potent siRNAs, thus limiting the ability of siRNAs with lower potency to mediate mRNA degradation. In contrast, a siRNA with low potency cannot compete with a highly efficient siRNA. We established a correlation between siRNA potency and ability to compete with other siRNAs. Thus, siRNAs that are more potent inhibitors for mRNA destruction have the potential to out-compete less potent siRNAs indicating that the amount of a cellular component, perhaps RISC, limits siRNA activity.

Binding, Competitive↗

PubMed2005

Regioselective syntheses of 7-nitro-7-deazapurine nucleosides and nucleotides for efficient PCR incorporation.

Substitution at the C(7) position of purine nucleotides by a potent electron-withdrawing nitro group facilitates the cleavage of glycosidic bonds under alkaline conditions. This property is useful for sequence-specific cleavage of DNA containing these analogues. Here we describe the preparation of 7-deaza-7-NO(2)-dA and 7-deaza-7-NO(2)-dG using two different approaches, starting from 2'-deoxy-adenosine and 6-chloro-7-deaza-guanine, respectively. These modified nucleosides were converted to nucleotide triphosphates, each of which can replace the corresponding, naturally occurring triphosphate to support PCR amplification. [structure: see text]

DNA↗

PubMed2005

Positional effect of chemical modifications on short interference RNA activity in mammalian cells.

A systematic study on the effect of 2'-sugar modifications (2'-F (2'-F-2'-deoxy-nucleoside residues), 2'-O-Me (2'-O-methyl-nucleoside residues), and 2'-O-MOE [2'-O-(2-methoxyethyl)]-nucleoside residues) in the antisense and sense strands of short interference RNA (siRNA) was performed in HeLa cells. The study of the antisense strand of siRNAs demonstrated that activity depends on the position of the modifications in the sequence. The siRNAs with modified ribonucleotides at the 5'-end of the antisense strand were less active relative to the 3'-modified ones. The 2'-F sugar was generally well-tolerated on the antisense strand, whereas the 2'-O-Me showed significant shift in activity depending on the position of modification. The 2'-O-MOE modification in the antisense strand resulted in less active siRNA constructs regardless of placement position in the construct. The incorporation of the modified residues, e.g., 2'-O-Me and 2'-O-MOE, in the sense strand of siRNA did not show a strong positional preference. These results may provide guidelines to design effective and stable siRNAs for RNA interference mediated therapeutic applications.

Animals↗

PubMed2005

Fully 2'-modified oligonucleotide duplexes with improved in vitro potency and stability compared to unmodified small interfering RNA.

We have identified a small interfering RNA (siRNA) motif, consisting entirely of 2'-O-methyl and 2'-fluoro nucleotides, that displays enhanced plasma stability and increased in vitro potency. At one site, this motif showed remarkable >500-fold improvement in potency over the unmodified siRNA. This marks the first report of such a potent fully modified motif, which may represent a useful design for therapeutic oligonucleotides.

Animals↗

PubMed2004

Structure-activity relationship of purine ribonucleosides for inhibition of hepatitis C virus RNA-dependent RNA polymerase.

As part of a continued effort to identify inhibitors of hepatitis C viral (HCV) replication, we report here the synthesis and evaluation of a series of nucleoside analogues and their corresponding triphosphates. Nucleosides were evaluated for their ability to inhibit HCV RNA replication in a cell-based, subgenomic replicon system, while nucleoside triphosphates were evaluated for their ability to inhibit in vitro RNA synthesis mediated by the HCV RNA-dependent RNA polymerase, NS5B. 2'-C-Methyladenosine and 2'-C-methylguanosine were identified as potent inhibitors of HCV RNA replication, and the corresponding triphosphates were found to be potent inhibitors of HCV NS5B-mediated RNA synthesis. The data generated in the cell-based assay demonstrated a fairly stringent structure-activity relationship around the active nucleosides. Increase in steric bulk beyond methyl on C2, change in the stereo- or regiochemistry of the methyl substituent, or change of identity of the heterobase beyond that of the endogenous adenine or guanine was found to lead to loss of inhibitory activity. The results highlight the importance of the ribo configuration 2'- and 3'-hydroxy pharmacophores for inhibition of HCV RNA replication in the cell-based assay and demonstrate that inclusion of the 2'-C-methylribonucleoside pharmacophore leads to increased resistance to adenosine deaminase and purine nucleoside phosphorylase mediated metabolism.

Adenosine Deaminase↗

PubMed2003

A high-capacity RNA affinity column for the purification of human IRP1 and IRP2 overexpressed in Pichia pastoris.

Regulated expression of proteins involved in mammalian iron metabolism is achieved in part through the interaction of the iron regulatory proteins IRP1 and IRP2 with highly conserved RNA stem-loop structures, known as iron-responsive elements (IREs), that are located within the 5' or 3' untranslated regions of regulated transcripts. As part of an effort to determine the structures of the IRP-IRE complexes using crystallographic methods, we have developed an efficient process for obtaining functionally pure IRP1 and IRP2 that relies upon the improved overexpression (>10 mg of soluble IRP per liter of culture) of each human IRP in the yeast Pichia pastoris and large-scale purification using RNA affinity chromatography. Despite the utility of RNA affinity chromatography in the isolation of RNA-binding proteins, current methods for preparing RNA affinity matrices produce columns of low capacity and limited stability. To address these limitations, we have devised a simple method for preparing stable, reusable, high-capacity RNA affinity columns. This method utilizes a bifunctional linker to covalently join a 5'-amino tethered RNA with a thiol-modified Sepharose, and can be used to load 150 nmole or more of RNA per milliliter of solid support. We demonstrate here the use of an IRE affinity column in the large-scale purification of IRP1 and IRP2, and suggest that the convenience of this approach will prove attractive in the analysis of other RNA-binding proteins.

Chromatography, Affinity↗