The concept of conformational restriction leading to the preorganization of modified strands has proven to be successful and has afforded nucleic acid analogues with many interesting properties suitable for various biochemical applications. We utilized this concept to prepare a set of constrained oligonucleotides derived from 1,4-dioxane and 1,3-dioxolane-locked nucleoside phosphonates and evaluated their hybridization affinities towards their complementary RNA strands. With an increase of ΔTmper modification up to +5.2 °C, the hybridization experiments revealed the (S)-2',3'-O-phosphonomethylidene internucleotide linkage as one of the most Tm-increasing modifications reported to date. Moreover, we introduced a novel prediction tool for the pre-selection of potentially interesting chemical modifications of oligonucleotides.
To determine the influence of internucleotide linkage and sugar ring conformation, and the role of 5'-terminal phosphate, on the activation of human RNase L, a series of 2'- and 5'-O-methylphosphonate-modified tetramers were synthesized from appropriate monomeric units and evaluated for their ability to activate human RNase L. Tetramers pAAAp(c)X modified by ribo, arabino or xylo 5'-phosphonate unit p(c)X activated RNase L with efficiency comparable to that of natural activator. Moreover, incorporation of phosphonate linkages ensured the stability against cleavage by nucleases. The substitution of 5'-terminal phosphate for 5'-terminal phosphonate in tetramer p(c)XAAA afforded tetramers with excellent activation efficiency and with complete stability against cleavage by phosphomonoesterases.
- MeSH
- adeninnukleotidy chemie MeSH
- bezbuněčný systém MeSH
- časové faktory MeSH
- chemické modely MeSH
- dimerizace MeSH
- endoribonukleasy chemie MeSH
- farmaceutická chemie metody MeSH
- kyseliny fosforité chemie MeSH
- lidé MeSH
- myši MeSH
- oligoribonukleotidy chemie MeSH
- racionální návrh léčiv MeSH
- rezonanční přenos fluorescenční energie metody MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Solid phase synthesis of phosphonate-modified oligoribonucleotides using 2'-O-benzoyloxymethoxymethyl protected monomers is presented in both 3'→5' and 5'→3' directions. Hybridisation properties and enzymatic stability of oligoribonucleotides modified by regioisomeric 3'- and 5'-phosphonate linkages are evaluated. The introduction of the 5'-phosphonate units resulted in moderate destabilisation of the RNA/RNA duplexes (ΔT(m)-1.8 °C/mod.), whereas the introduction of the 3'-phosphonate units resulted in considerable destabilisation of the duplexes (ΔT(m)-5.7 °C/mod.). Molecular dynamics simulations have been used to explain this behaviour. Both types of phosphonate linkages exhibited remarkable resistance in the presence of ribonuclease A, phosphodiesterase I and phosphodiesterase II.
- MeSH
- exonukleasy metabolismus MeSH
- fosfodiesterasa I metabolismus MeSH
- kyseliny fosforité chemická syntéza chemie metabolismus MeSH
- oligoribonukleotidy chemická syntéza chemie metabolismus MeSH
- pankreatická ribonukleasa metabolismus MeSH
- simulace molekulární dynamiky MeSH
- techniky syntézy na pevné fázi MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
To determine the influence of methylene group insertion in the internucleotide linkage on the binding process of 2',5'-oligoadenylates to RNase L, a series of 2'-phosphonate-modified trimers and tetramers were synthesized from appropriate monomeric units and evaluated for their ability to bind to murine RNase L. Tetramers pAAXA modified by ribo-, arabino-, or xylo-2'-phosphonate unit X in the third position were capable of binding to RNase L in nanomolar concentrations. The replacement of the first residue (pXAAA), or both the first and the third residues (pXAXA), was also tolerated by the enzyme. In contrast, in all cases, the replacement of the second residue (pAXAA) resulted in the significant decrease of binding ability. Additionally, no more than two phosphonate modifications in the tetramer were allowed to retain the binding affinity to the enzyme. Although all three tetramers pAAXA were found to be potent enzyme binders, only tetramers modified by ribo- and xylo-2'-phosphonate unit X activated the RNase L-catalyzed cleavage of the RNA substrate. Surprisingly, tetramer pAAXA, modified by arabino-2'-phosphonate unit X, did not activate the enzyme and can be considered a potent antagonist. In comparison with their natural counterpart, the phosphonate analogues of the pA4 exhibit superior resistance toward nucleases present in the murine spleen homogenate.
- MeSH
- adeninnukleotidy farmakologie chemická syntéza chemie MeSH
- endoribonukleasy metabolismus MeSH
- financování organizované MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- oligoribonukleotidy farmakologie chemická syntéza chemie MeSH
- organofosfonáty farmakologie chemická syntéza chemie MeSH
- slezina enzymologie MeSH
- stereoizomerie MeSH
- techniky in vitro MeSH
- vazba proteinů MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
