modified oligonucleotide
Dotaz
Zobrazit nápovědu
- MeSH
- amfotericin B farmakologie MeSH
- buněčná membrána metabolismus MeSH
- buněčné jádro metabolismus MeSH
- cytoplazma metabolismus MeSH
- finanční podpora výzkumu jako téma MeSH
- fluorescenční spektrometrie metody MeSH
- kationty MeSH
- myši MeSH
- oligonukleotidy farmakokinetika MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
Three structurally diverse types of the protected pyrrolidine nucleoside phosphonates were prepared as the monomers for the introduction of pyrrolidine nucleotide units into modified oligonucleotides on the solid phase. Two different chemistries were used for incorporation of modified and natural units: the phosphotriester method for the former, i.e., monomers containing N-phosphonoalkyl and N-phosphonoacyl moieties attached to the pyrrolidine ring nitrogen atom, and phosphoramidite chemistry for the latter. Since the synthesized pyrrolidine nucleoside phosphonic acids are close mimics of the 3'-deoxynucleoside 5'-phosphates, the incorporation of one modified unit into oligonucleotides gives rise to one 2',5' internucleotide linkage. A series of nonamers containing two or three modified units, as well as the fully modified adenine 15-mer, were synthesized in reverse order, i.e., from the 5' to the 3' end of the strand. The measurement of thermal characteristics of the complexes of modified nonamers with the complementary strand revealed a destabilizing effect of the introduced modification. The modified adenine homooligonucleotide, was found to form the most stable complex with oligothymidylate of all the tested modified oligonucleotides in terms of ?Tm per modification.
Retroviral integrase participates in two catalytic reactions, which require interactions with the two ends of the viral DNA in the 3'processing reaction, and with a targeted host DNA in the strand transfer reaction. The 3'-hydroxyl group of 2'-deoxyadenosine resulting from the specific removing of GT dinucleotide from the viral DNA in the processing reaction provides the attachment site for the host DNA in a transesterification reaction. We synthesized oligonucleotides (ONs) of various lengths that mimic the processed HIV-1 U5 terminus of the proviral long terminal repeat (LTR) and are ended by 2'-deoxyadenosine containing a 3'-O-phosphonomethyl group. The duplex stability of phosphonomethyl ONs was increased by covalent linkage of the modified strand with its complementary strand by a triethylene glycol loop (TEG). Modified ONs containing up to 10 bases inhibited in vitro the strand transfer reaction catalyzed by HIV-1 integrase at nanomolar concentrations.
- MeSH
- financování organizované MeSH
- HIV - dlouhá koncová repetice genetika MeSH
- HIV-integrasa účinky léků MeSH
- inhibitory HIV-integrasy farmakologie chemická syntéza MeSH
- konformace nukleové kyseliny MeSH
- kyseliny fosforu MeSH
- molekulární mimikry MeSH
- oligonukleotidy farmakologie chemická syntéza MeSH
Enzymatic synthesis of short (10-22 nt) base-modified oligonucleotides (ONs) was developed by nicking enzyme amplification reaction (NEAR) using Vent(exo-) polymerase, Nt.BstNBI nicking endonuclease, and a modified deoxyribonucleoside triphosphate (dNTP) derivative. The scope and limitations of the methodology in terms of different nucleobases, length, sequences, and modifications has been thoroughly studied. The methodology including isolation of the modified ONs was scaled up to nanomolar amounts and the modified ONs were successfully used as primers in primer extension and PCR. Two simple and efficient methods for fluorescent labeling of the PCR products were developed, based either on direct fluorescent labeling of primers or on NEAR synthesis of ethynylated primers, PCR, and final click labeling with fluorescent azides.
- MeSH
- azidy chemie MeSH
- click chemie MeSH
- deoxyribonukleotidy chemická syntéza chemie metabolismus MeSH
- DNA primery biosyntéza genetika MeSH
- endonukleasy metabolismus MeSH
- fluorescenční barviva chemie MeSH
- molekulární struktura MeSH
- oligonukleotidy biosyntéza MeSH
- polymerázová řetězová reakce * MeSH
- techniky amplifikace nukleových kyselin * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Protected N-branched nucleoside phosphonates containing adenine and thymine bases were prepared as the monomers for the introduction of aza-acyclic nucleotide units into modified oligonucleotides. The phosphotriester and phosphoramidite methods were used for the incorporation of modified and natural units, respectively. The solid phase synthesis of a series of nonamers containing one central modified unit was successfully performed in both 3'→5' and 5'→3' directions. Hybridization properties of the prepared oligoribonucleotides and oligodeoxyribonucleotides were evaluated. The measurement of thermal characteristics of the complexes of modified nonamers with the complementary strand revealed a considerable destabilizing effect of the introduced units. We also examined the substrate/inhibitory properties of aza-acyclic nucleoside phosphono-diphosphate derivatives (analogues of nucleoside triphosphates) but neither inhibition of human and bacterial DNA polymerases nor polymerase-mediated incorporation of these triphosphate analogues into short DNA was observed.
- MeSH
- adenin chemická syntéza chemie MeSH
- DNA-dependentní DNA-polymerasy metabolismus MeSH
- inhibitory syntézy nukleových kyselin chemická syntéza chemie farmakologie MeSH
- lidé MeSH
- nukleosidy chemická syntéza chemie farmakologie MeSH
- oligonukleotidy chemická syntéza chemie farmakologie MeSH
- organofosfonáty chemická syntéza chemie farmakologie MeSH
- sekvence nukleotidů MeSH
- thymin chemická syntéza chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
