ABSTRACT
Powerful pyrene probes: Two kinds of pyrene-labeled oligonucleotides (HNA- and RNA-skeleton probes) were explored. The enhanced fluorescence intensity in the monomer region and the disappearance of aggregate/excimer emission in duplexes has been successfully used to detect the hybridization of oligonucleotides. By covalently attaching pyrene chromophores with different linkers onto altritol nucleotides or ribonucleotides, and by varying the number of these pyrene modified altritol nucleotides and ribonucleotides in HNA (hexitol nucleic acid) and RNA, respectively, we have explored the general applicability of pyrene absorbance and especially fluorescence as a probe to monitor RNA hybridization. The results reveal that the backbone of the probes, the number of pyrene units attached and the nature of the tether can all substantially affect the absorbance and fluorescence properties of the probes both in single strand and double strand form. Moreover, the strength of hybridization is also affected. The disappearance of pyrene aggregate/excimer emission and simultaneous increase in monomer emission intensity of the multipyrene-labeled probes has been successfully used to monitor the hybridization of oligonucleotides, including a hairpin structure. Differences in optical response between the HNA- and RNA-skeleton probes upon hybridization indicate that the interaction of pyrene with the nucleobases in both types of duplexes is different.
Subject(s)
Oligonucleotide Probes/chemistry , Pyrenes/chemistry , RNA/chemistry , Fluorescent Dyes/chemistry , Nucleic Acid Hybridization , Oligonucleotide Probes/chemical synthesis , Pyrenes/chemical synthesis , Spectrometry, Fluorescence , Temperature , Transition TemperatureABSTRACT
The use of chemically synthesized short interfering RNAs (siRNAs) is currently the method of choice to manipulate gene expression in mammalian cell culture, yet improvements of siRNA design is expectably required for successful application in vivo. Several studies have aimed at improving siRNA performance through the introduction of chemical modifications but a direct comparison of these results is difficult. We have directly compared the effect of 21 types of chemical modifications on siRNA activity and toxicity in a total of 2160 siRNA duplexes. We demonstrate that siRNA activity is primarily enhanced by favouring the incorporation of the intended antisense strand during RNA-induced silencing complex (RISC) loading by modulation of siRNA thermodynamic asymmetry and engineering of siRNA 3'-overhangs. Collectively, our results provide unique insights into the tolerance for chemical modifications and provide a simple guide to successful chemical modification of siRNAs with improved activity, stability and low toxicity.
Subject(s)
RNA Interference , RNA, Small Interfering/chemistry , Cell Line, Tumor , Cell Survival , Humans , RNA Stability , RNA, Small Interfering/blood , RNA, Small Interfering/toxicity , RNA-Induced Silencing Complex/metabolismABSTRACT
A simple and efficient method for the preparation of pyrimidine 2'-O-hydroxyethoxymethylribonucleosides and 2'-O-hydroxypropoxymethylribonucleosides has been developed. These modified nucleosides were incorporated into oligoribonucleotides, which were shown to form stable RNA/RNA duplexes. The effect of 2' -O-modification in the antisense and sense strands of small interference RNA was evaluated in multi-drug resistant NIH 3T3 cells.
Subject(s)
Oligoribonucleotides/chemistry , Pyrimidines/chemistry , RNA/chemistry , Ribonucleosides/chemical synthesis , Ribonucleosides/chemistryABSTRACT
A simple and efficient method for the preparation of 2-O-beta-D-ribofuranosylnucleosides, minor tRNA components, is described in this unit. The method consists of condensation of a small excess of 1-O-acetyl-2,3,5-tri-O-benzoyl-beta-D-ribofuranose activated with tin tetrachloride with N-protected 3,5-O-tetra-isopropyldisiloxane-1,3-diyl-ribonucleosides in 1,2-dichloroethane. Subsequent deprotection produces 2-O-beta-D-ribofuranosylnucleosides in an overall yield of 46% to 72%.
Subject(s)
Furans/chemistry , Ribonucleosides/chemical synthesis , Chromatography, Thin Layer , Magnetic Resonance Spectroscopy , Ribonucleosides/chemistryABSTRACT
The formation of a disaccharide nucleoside (11) by O3'-glycosylation of 5'-O-protected 2'-deoxyadenosine or its N6-benzoylated derivative has been observed to be accompanied by anomerisation to the corresponding alpha-anomeric product (12). The latter reaction can be explained by instability of the N-glycosidic bond of purine 2'-deoxynucleosides in the presence of Lewis acids. An independent study on the anomerisation of partly blocked 2'-deoxyadenosine has been carried out. Additionally, transglycosylation has been utilized in the synthesis of 3'-O-beta-D-ribofuranosyl-2'deoxyadenosines and its alpha-anomer.
