34 research outputs found
Strategies for Oligoribonucleotide Synthesis According to the Phosphoramidite Method
Advances in oligoribonucleotide synthesis have lagged behind those in oligodeoxyribonucleotide synthesis because of the difficulty in identifying orthogonal protecting groups for the 2â˛â and 5â˛âhydroxyls. Adaptation of the phosphoramidite method for DNA synthesis to RNA synthesis has greatly improved our understanding of RNA. It allows siteâspecific introduction of modified nucleosides to any position in an RNA molecule, as well as introduction of variations at multiple sites in the molecule. This overview discusses issues that are relevant to RNA synthesis by the phosphoramidite approach, including supports used, activation of the ribonucleoside phosphoramidites, and protection of the nucleobase, phosphate, and 2â˛â and 5â˛âhydroxyls.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143793/1/cpnc0305.pd
Change in MRI synovitis correlates with change in pain following intra-articular steroid injection
Rapid synthesis of oligodeoxyribonucleotides III. Effect of added carboxylate ion on the efficiency of internucleotide bond formation. Solid-phase synthesis of the dodecanucleotide, d(pT-A-A-C-T-G-C-T-C-A-C-T)
Synthesis of Branched DNA Using Oxidatively Cleavable Tritylsulfenyl as a Hydroxy Protecting Group
MALDI-TOF mass spectral analysis of siRNA degradation in serum confirms an RNAse A-like activity
Synthetic siRNA duplexes are used widely as reagents for silencing of mRNA targets in cells and are being developed for in vivo use. Serum stability is a major concern if siRNA is to be used for therapeutic delivery within blood circulation. We have developed the use of MALDI-TOF mass spectrometry as a rapid and convenient analytical tool to identify the most vulnerable sites within siRNA to serum degradation. Using this approach, we found that one siRNA duplex (Dh3) with UpA sequences close to one end was particularly vulnerable to rapid cleavage. This produced a fragment of mass consistent with the presence of a 2â˛,3â˛-cyclic phosphate that was slowly hydrolysed to a 2â˛-(3â˛-)phosphate on extended incubation. Substitution of these sites with 2â˛-O-methyl U residues prevented cleavage and confirmed that the major pathway for initial degradation is via cleavage by an RNAse A-like activity. Mass spectral analysis was used to follow the serum degradation of siRNA over more prolonged periods to show the accumulation of many fragments, almost all showing cleavage following pyrimidine nucleoside residues. Overall, the MALDI-TOF mass spectral analysis technique should prove useful for preliminary screening of the serum stability of siRNA duplexes and for identification of the most vulnerable cleavage sites
RNA Synthesis by Reverse Direction Process: Phosphoramidites and High Purity RNAs and Introduction of Ligands, Chromophores, and Modifications at 3â˛âEnd
In vitro and in vivo delivery studies of siRNA-peptide conjugates
siRNA is a novel reagent for targeting RNA in cells and reducing gene expression. However, its major limitation is the need for a suitable carrier for specific delivery into cell cultures or animal tissues without associated toxicity. In the work presented here, we use different cell penetrating peptides (CPPs) for in vitro and in vivo delivery of various siRNAs in the absence of other transfection reagents. CPPs were conjugated to the siRNA sense strand (OMe/DNA or RNA) by a disulfide linkage and subsequently hybridized to the complementary antisense RNA strand. Amongst the CPPs studied were Penetratin, Transportan and Tat (residues 48-58). CPP-siRNA conjugates were evaluated initially using either an in vitro model targeting plasmid-encoded firefly luciferase or by targeting a disease-relevant endogenous gene coding for p38 a MAP kinase. The ability to modulate levels of endogenous MAPK14 (p38 a ) expression is important in validating the role of the p38 pathway in disease models and in establishing new drug targets. The results showed that free CPP-5â-siRNA (Luc) conjugates were unable to knockdown firefly luciferase expression at low concentrations (up to 500 nM), when analyzed by luminescence 24 hr post-transfection in HeLa or HepG2 cells. In contrast, it was found that certain CPP-siRNA(p38) conjugates were able to knockdown p38 a MAP kinase expression. HeLa cells were incubated with CPP-siRNA(p38) conjugates in serum-free media for 24 hr and p38 expression normalised to 18S rRNA was determined by quantitative real-time PCR using Taqman probes. Following 24 hr transfection, p38 knockdown was achieved with CPP 5â- and 3â-conjugated siRNA. The highest activity was observed for the Penetratin 3â-conjugate, which gave ~ 50% knockdown (P < 0.01) of p38 expression (n=4) at 10 ÂľM. Certain CPP-siRNA conjugates were evaluated in an in vivo mouse model, by targeting p38 a MAP kinase in lungs following intratracheal instillation. p38 mRNA knockdown efficiency, duration and siRNA recovery from the mouse lung appeared to be dose-dependent over a 24-hour period in the absence of delivery reagents/CPPs. Conjugation of Penetratin or Tat did not appear to improve significantly upon knockdown efficiency. Whilst recovery of siRNA from lung homogenates was impaired due to CPP conjugation, our findings suggest that the knockdown efficiency paradox compared to our in vitro data may relate to sequence-dependent siRNA instability