Two hairpin hexa(N-methylpyrrole)carboxamide DNA minor groove binders (MGB) were linked together via their N-termini in head-to-head orientation. Complex formation between these bis-MGB conjugates ...and target DNA has been studied using DNase I footprint- ing, circular dichroism, thermal dissociation, and molecular modeling. DNase I footprint revealed binding of these conjugates to all the sites of 492 b.p. DNA fragment containing (A/T)
n
X
m
(A/T)
p
sequences, where n>3, p>3; m=1,2; X = A,T,G, or C. Binding affinity depended on the sequence context of the target. CD experiments and molecular modeling showed that oligo(N-methylpyrrole)carboxamide moieties in the complex form two short antiparallel hairpins rather than a long parallel head-to-head hairpin. Binding of bis-MGB also stabilized a target duplex thermodynamically. Sequence specificity of bis-MGB/DNA binding was validated using bis-conjugates of sequence-specific hairpin (N-methylpyrrole)/(N-methylimidazole) carboxamides.
In order to increase the size of recognition sequence, the conjugates of bis-MGB with triplex-forming oligonucleotides (TFO) were synthesized and compared to TFO conjugated with single MGB hairpin unit. Bis-MGB-oligonucleotide conjugates also bind to two blocks of three and more A·T/T·A pairs similarly to bis-MGB alone, independently of the oligonucleotide moiety, but with lower affinity. However, the role of TFO in DNA recognition was demonstrated for mono-MGB-TFO conjugate where the binding was detected mainly in the area of the target sequence consisting of both MGB and TFO recognition sites.
Basing on the molecular modeling, three-dimensional models of both target DNA/bis-MGB and target DNA/TFO-bis-MGB complexes were built, where bis-MGB forms two antiparallel hairpins. According to the second model, one MGB hairpin is in the minor groove of 5′-adjacent A/T sequence next to the triplex-forming region, whereas the other one occupies the minor groove of the TFO binding polypurine tract. All these data together give a key information for the construction of MGB-MGB and MGB-oligonucleotide conjugates possessing high specificity and affinity for the target double-stranded DNA.
We have developed a new method for the preparation of oligodeoxyribonucleotides and oligo(2′-O-methylribonucleotides) that contain a 2′-phosphorylated ribonucleoside residue, and optimized it to ...avoid 2′ -3′ -isomerization and chain cleavage. Structures of the 2′ -phosphorylated oligonucleotides were confirmed by MALDI-TOF MS and enzymatic digestion, and the stability of their duplexes with DNA and RNA was investigated. 2′-Phosphorylated oligonucleotides may be useful intermediates for the introduction of various chemical groups for a wide range of applications.
Abstract
We designed tandems of oligo(2′-O-methylribonucleotides) conjugates containing two bispyrene (5′-bisPyr and 3′-bisPyr) groups on their junction for detection of C677T polymorphism in the ...methylenetetrahydrofolate reductase gene (MHTFR). The potential of SNP detection with multipyrene tandems of oligo(2′-O-methylribonucleotides) was demonstrated.
New eximer-based tandem systems for SNP detection Novopashina, Daria S; Meschaninova, Maria I; Kholodar, Svetlana A ...
Nucleic Acids Symposium Series,
2008, Letnik:
52, Številka:
1
Journal Article
Odprti dostop
Abstract
We report here the design and synthesis of new mono- and bis-pyrene-labeled oligo(2′-Omethylribonucleotide) tandems as perspective probes for SNP detection. The detection strategy is based ...on the eximer formation when two or more pyrene groups are brought into close proximity upon hybridization of the tandem components with DNA. The potential of SNP detection with tandems of pyrene-labeled oligo(2′-O-methylribonucleotides) by duplex melting curve analysis based on excimer fluorescence registration was demonstrated.
The thermodynamic asymmetry of siRNA duplexes determines their silencing activity. Favorable asymmetry can be achieved by incorporation of mismatches into the 3' part of the sense strand, providing ...fork-siRNAs, which exhibit higher silencing activity and higher sensitivity to nucleases. Recently, we found that selective 2'-O-methyl modifications of the nuclease-sensitive sites of siRNA significantly improve its nuclease resistance without substantial loss of silencing activity. Here, we examined the impact of nucleotide mismatches and the number and location of 2'-O-methyl modifications on the silencing activity and nuclease resistance of anti-MDR1 siRNAs. We found that both nonmodified and selectively modified fork-siRNAs with 4 mismatches at the 3' end of the sense strand suppress the expression of target gene at lower effective concentrations than the parent siRNAs with classical duplex design. The selective modification of nuclease-sensitive sites significantly improved the stability of fork-siRNAs in the presence of serum. The selectively modified fork-siRNA duplexes provided inhibitory effect over a period of 12 days posttransfection, whereas the gene silencing activity of the nonmodified analogs expired within 6 days. Thus, selective chemical modifications and structural alteration of siRNA duplexes improve their silencing properties and significantly prolong the duration of their silencing effect.
PDF
