We designed and synthesized a series of 2'-deoxyribonucleoside triphosphates (dNTPs) bearing various lipid moieties. Fatty acid- and cholesterol-modified dNTPs proved to be substrates for KOD XL DNA polymerase in primer extension reactions. They were also mutually compatible for simultaneous multiple incorporations into the DNA strand. The methodology of enzymatic synthesis opened a pathway to diverse structurally unique lipid-ON probes containing one or more lipid units. We studied interactions of such probes with the plasma membranes of live cells. Employing a rational design, we found a series of lipid-ONs with enhanced membrane anchoring efficiency. The in-membrane stability of multiply modified ONs was superior to that of commonly studied ON analogues, in which a single cholesterol molecule is typically tethered to the thread end. Notably, some of the probes were detected at the cell surface even after 24 h upon removal of the probe solution. Such an effect was general to several studied cell lines.

• Publikační typ
• časopisecké články MeSH

A set of modified 2'-deoxyribonucleoside triphosphates (dNTPs) bearing a linear or branched alkane, indole or phenyl group linked through ethynyl or alkyl spacer were synthesized and used as substrates for polymerase synthesis of hypermodified DNA by primer extension (PEX). Using the alkyl-linked dNTPs, the polymerase synthesized up to 22-mer fully modified oligonucleotide (ON), whereas using the ethynyl-linked dNTPs, the enzyme was able to synthesize even long sequences of >100 modified nucleotides in a row. In PCR, the combinations of all four modified dNTPs showed only linear amplification. Asymmetric PCR or PEX with separation or digestion of the template strand can be used for synthesis of hypermodified single-stranded ONs, which are monodispersed polymers displaying four different substituents on DNA backbone in sequence-specific manner. The fully modified ONs hybridized with complementary strands and modified DNA duplexes were found to exist in B-type conformation (B- or C-DNA) according to CD spectral analysis. The modified DNA can be replicated with high fidelity to natural DNA through PCR and sequenced. Therefore, this approach has a promising potential in generation and selection of hypermodified aptamers and other functional polymers.

• MeSH
• adenin chemie   metabolismus   MeSH
• aptamery nukleotidové chemická syntéza   genetika   MeSH
• cytosin chemie   metabolismus   MeSH
• deoxyribonukleosidy chemie   genetika   metabolismus   MeSH
• dinukleosidfosfáty chemie   genetika   metabolismus   MeSH
• DNA-dependentní DNA-polymerasy genetika   metabolismus   MeSH
• DNA chemie   genetika   metabolismus   MeSH
• guanin chemie   metabolismus   MeSH
• hydrofobní a hydrofilní interakce MeSH
• párování bází MeSH
• polymerázová řetězová reakce MeSH
• polymery chemická syntéza   metabolismus   MeSH
• replikace DNA * MeSH
• sekvence nukleotidů MeSH
• uracil chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenin MeSH
• aptamery nukleotidové MeSH
• cytosin MeSH
• deoxyribonukleosidy MeSH
• dinukleosidfosfáty MeSH
• DNA-dependentní DNA-polymerasy MeSH
• DNA MeSH
• guanin MeSH
• polymery MeSH
• uracil MeSH