Nucleic acid aptamers are single-stranded (ss)DNA or RNA oligonucleotides that can take various conformations and bind specifically and with high affinity to selected targets. While the introduction of SELEX (systematic evolution of ligands by exponential enrichment) revolutionized the production of the aptamers, this procedure is impeded by the formation of undesirable by-products reflecting hybridization among complementary oligonucleotides in the ssDNA libraries during asymmetric PCR. To reduce nonspecific amplification we tested cellulose-derived compounds and found that sodium carboxymethylcellulose (CMC) at a concentration 0.05%-0.2% efficiently suppressed production of undesirable large DNA amplicons during asymmetric PCR in the course of SELEX. Formation of the PCR by-products was reduced by CMCs of low and medium viscosity more than by CMCs of high viscosity, and all of them bound to DNA oligonucleotides as determined by electrophoresis in agarose gels. In contrast to CMC, methylcellulose did not reduce the formation of the PCR by-products and did not bind to DNA. DNA aptamers selected in the presence of CMC could be used directly in enzyme-linked immunosorbent-like assay. The combined data suggest that CMC binds weekly to DNA oligonucleotides through hydroxyl groups and in this way inhibits low-affinity DNA-DNA hybridization and enhances the production of specific amplicons in asymmetric PCR.

• Klíčová slova
• Carboxymethylcellulose, DNA aptamers, ELISA, PCR, Single-stranded DNA,
• MeSH
• aptamerová technika SELEX metody   MeSH
• aptamery nukleotidové chemie   MeSH
• ELISA metody   MeSH
• jednovláknová DNA chemie   MeSH
• methylcelulosa chemie   MeSH
• polymerázová řetězová reakce metody   MeSH
• sodná sůl karboxymethylcelulosy chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aptamery nukleotidové MeSH
• jednovláknová DNA MeSH
• methylcelulosa MeSH
• sodná sůl karboxymethylcelulosy MeSH

Single-stranded model oligodeoxyribonucleotides, each containing a single protonatable base-cytosine, adenine, guanine, or 5-methylcytosine-centrally located in a background of non-protonatable thymine residues, were acid-titrated in aqueous solution, with UV monitoring. The basicity of the central base was shown to depend on the type of the central base and its nearest neighbours and to rise with increasing oligonucleotide length and decreasing ionic strength of the solution. More complex model oligonucleotides, each containing a centrally located 5-methylcytosine base, were comparatively evaluated in single-stranded and double-stranded form, by UV spectroscopy and high-field NMR. The N3 protonation of the 5-methylcytosine moiety in the double-stranded case occurred at much lower pH, at which the duplex was already experiencing general dissociation, than in the single-stranded case. The central guanine:5-methylcytosine base pair remained intact up to this point, possibly due to an unusual alternative protonation on O2 of the 5-methylcytosine moiety, already taking place at neutral or weakly basic pH, as indicated by UV spectroscopy, thus suggesting that 5-methylcytosine sites in double-stranded DNA might be protonated to a significant extent under physiological conditions.

• Klíčová slova
• DNA methylation, methylcytosine, nucleobases, oligonucleotides, protonation,
• MeSH
• 5-methylcytosin metabolismus   MeSH
• adenin metabolismus   MeSH
• guanin metabolismus   MeSH
• jednovláknová DNA * chemie   metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• konformace nukleové kyseliny MeSH
• oligodeoxyribonukleotidy * chemie   metabolismus   MeSH
• osmolární koncentrace MeSH
• protony MeSH
• sekvence nukleotidů MeSH
• thymin metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• 5-methylcytosin MeSH
• adenin MeSH
• guanin MeSH
• jednovláknová DNA * MeSH
• oligodeoxyribonukleotidy * MeSH
• protony MeSH
• thymin MeSH

Earlier, we showed that using differential pulse cathodic stripping voltammetry with hanging mercury drop electrode (HMDE), single-stranded (ss) DNA modified with osmium tetroxide, pyridine reagent (Os,py) can be determined at concentrations down to about 10-5 ng/ml. Here, we show that by exchanging Os,py for osmium tetroxide, 2,2'-bipyridine (Os,bipy) and decreasing the pH of the background electrolyte from neutrality to about pH 4, ssDNA can be determined at concentrations lower by one order of magnitude. Determination of DNA at such low concentrations may find use in various areas of molecular biology and in biotechnologies, including the development of DNA sensors.

• MeSH
• elektrochemie metody   MeSH
• jednovláknová DNA analýza   chemie   MeSH
• osmium chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• jednovláknová DNA MeSH
• osmium MeSH

Interactions of echinomycin (Echi) with DNA was studied by cyclic voltammetry (CV) with hanging mercury drop electrode (HMDE). Echinomycin was electrochemically active, yielding several signals. Interaction of Echi with dsDNA attached to a hanging mercury drop electrode resulted in high Echi signals, suggesting a strong binding of Echi to dsDNA by bis-intercalation at the electrode surface. Under the same conditions, interaction of Echi with ssDNA produced almost no Echi signal. This behavior is in agreement with a strong binding of Echi to dsDNA and a very weak binding of Echi to ssDNA observed earlier in solution. Echi, thus, appears to be a good candidate for redox indicator in electrochemical DNA hybridization sensors.

• MeSH
• DNA chemie   MeSH
• echinomycin chemie   MeSH
• elektrody * MeSH
• jednovláknová DNA chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• echinomycin MeSH
• jednovláknová DNA MeSH

RECQ5 is one of five RecQ helicases found in humans and is thought to participate in homologous DNA recombination by acting as a negative regulator of the recombinase protein RAD51. Here, we use kinetic and single molecule imaging methods to monitor RECQ5 behavior on various nucleoprotein complexes. Our data demonstrate that RECQ5 can act as an ATP-dependent single-stranded DNA (ssDNA) motor protein and can translocate on ssDNA that is bound by replication protein A (RPA). RECQ5 can also translocate on RAD51-coated ssDNA and readily dismantles RAD51-ssDNA filaments. RECQ5 interacts with RAD51 through protein-protein contacts, and disruption of this interface through a RECQ5-F666A mutation reduces translocation velocity by ∼50%. However, RECQ5 readily removes the ATP hydrolysis-deficient mutant RAD51-K133R from ssDNA, suggesting that filament disruption is not coupled to the RAD51 ATP hydrolysis cycle. RECQ5 also readily removes RAD51-I287T, a RAD51 mutant with enhanced ssDNA-binding activity, from ssDNA. Surprisingly, RECQ5 can bind to double-stranded DNA (dsDNA), but it is unable to translocate. Similarly, RECQ5 cannot dismantle RAD51-bound heteroduplex joint molecules. Our results suggest that the roles of RECQ5 in genome maintenance may be regulated in part at the level of substrate specificity.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• bodová mutace MeSH
• helikasy RecQ genetika   metabolismus   ultrastruktura   MeSH
• homologní rekombinace * MeSH
• hydrolýza MeSH
• jednovláknová DNA metabolismus   ultrastruktura   MeSH
• kinetika MeSH
• lidé MeSH
• mikroskopie atomárních sil MeSH
• missense mutace MeSH
• molekulární motory metabolismus   ultrastruktura   MeSH
• rekombinantní fúzní proteiny metabolismus   MeSH
• rekombinantní proteiny metabolismus   MeSH
• rekombinasa Rad51 genetika   metabolismus   MeSH
• replikační protein A metabolismus   MeSH
• substrátová specifita MeSH
• zobrazení jednotlivé molekuly * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• adenosintrifosfát MeSH
• helikasy RecQ MeSH
• jednovláknová DNA MeSH
• molekulární motory MeSH
• RAD51 protein, human MeSH Prohlížeč
• RECQL5 protein, human MeSH Prohlížeč
• rekombinantní fúzní proteiny MeSH
• rekombinantní proteiny MeSH
• rekombinasa Rad51 MeSH
• replikační protein A MeSH
• RPA1 protein, human MeSH Prohlížeč

A new, highly denaturing electrolyte system based on a solution containing 0.01 M NaOH, 0.0015 M Na2B4O5(OH)4 and a replaceable polymer sieving medium was designed for the separation of single-stranded DNA fragments in bare fused-silica capillaries. Extreme denaturing power, together with the optimized composition of the electrolyte, allows for a separation efficiency as high as 2,300,000 height equivalents to a theoretical plate per meter. Sample denaturation in alkaline solutions provides single-stranded DNA fragments without any intra- or intermolecular interactions at room temperature. Their electrophoretic mobilities were found to be twice those of fragments denatured by dimethylformamide or HCl. This can be interpreted in terms of an increased effective charge on the DNA molecules. The surprisingly weak electroosmosis (6 x 10(-10) m2 V-1 s-1) of polymer solutions at pH 12 or higher is considered to be the result of the dissolution of the silica capillary wall. A highly viscous thin layer of dissolved silica probably causes a shift of the slipping plane further away from the wall to the lower value of the zeta potential. Applications of the electrolyte in clinical diagnostics demonstrate its remarkable properties.

• MeSH
• denaturace nukleových kyselin MeSH
• elektroforéza kapilární metody   MeSH
• elektrolyty MeSH
• hydroxid sodný chemie   MeSH
• jednovláknová DNA chemie   izolace a purifikace   MeSH
• oxid křemičitý chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• elektrolyty MeSH
• hydroxid sodný MeSH
• jednovláknová DNA MeSH
• oxid křemičitý MeSH

Polyacrylamide gel electrophoresis is a widely used method to study short DNA fragments in solution. It is, however, a relative method requiring length markers to assess mobility, shape, flexibility, and molecularity of the DNA structures of interest. In recent literature we have encountered the use of oligo(dT) fragments as the native PAGE length markers. We show here that this practice is inadequate because oligo(dT) migration is strongly retarded in native polyacrylamide gels. This conclusion is qualitatively true irrespective of the conditions of electrophoresis, oligo(dT) length, and gel concentration. Depending on their length, oligo(dT) fragments migrate 2--4 times slower than that would correspond to their nucleotide number. This leads to erroneous conclusions, e.g., determination of the number of associated molecules in guanine quadruplexes or other DNA complexes.

• MeSH
• elektroforéza v polyakrylamidovém gelu metody   MeSH
• jednovláknová DNA izolace a purifikace   MeSH
• oligodeoxyribonukleotidy izolace a purifikace   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• jednovláknová DNA MeSH
• oligo (dT) MeSH Prohlížeč
• oligodeoxyribonukleotidy MeSH

THE 5'-exonucleases are enzymes that are essential for DNA replication and repair. As well as their exonucleolytic action, removing nucleotides from the 5'-end of nucleic acid molecules such as Okazaki fragments, many 5'-3'-exonucleases have been shown to possess endonucleolytic activities. T5 5'-3'-exonuclease shares many similarities with the amino terminal of eubacterial DNA polymerases, although, unlike eubacteria, phages such as T5, T4 and T7 express polymerase and 5'-exonuclease proteins from separate genes. Here we report the 2.5-A crystal structure of the phage T5 5'-exonuclease, which reveals a helical arch for binding DNA. We propose a model consistent with a threading mechanism in which single-stranded DNA could slide through the arch, which is formed by two helices, one containing positively charged, and the other hydrophobic, residues. The active site is at the base of the arch, and contains two metal-binding sites.

• MeSH
• elektrochemie MeSH
• exodeoxyribonukleasy chemie   metabolismus   MeSH
• jednovláknová DNA metabolismus   MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• povrchové vlastnosti MeSH
• T-fágy enzymologie   MeSH
• vazebná místa MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• exodeoxyribonukleasy MeSH
• jednovláknová DNA MeSH
• T5 D15 exonuclease MeSH Prohlížeč

Repetitive extragenic palindrome (REP)-associated tyrosine transposase enzymes (RAYTs) bind REP DNA domains and catalyze their cleavage. Genomic sequence analyses identify potential noncoding REP sequences associated with RAYT-encoding genes. To probe the conformational space of potential RAYT DNA binding domains, we report here spectroscopic and calorimetric measurements that detect and partially characterize the solution conformational heterogeneity of REP oligonucleotides from six bacterial species. Our data reveal most of these REP oligonucleotides adopt multiple conformations, suggesting that RAYTs confront a landscape of potential DNA substrates in dynamic equilibrium that could be selected, enriched, and/or induced via differential binding. Thus, the transposase-bound DNA motif may not be the predominant conformation of the isolated REP domain. Intriguingly, for several REPs, the circular dichroism spectra suggest guanine tetraplexes as potential alternative or additional RAYT recognition elements, an observation consistent with these REP domains being highly nonrandom, with tetraplex-favoring 5'-G and 3'-C-rich segments. In fact, the conformational heterogeneity of REP domains detected and reported here, including the formation of noncanonical DNA secondary structures, may reflect a general feature required for recognition by RAYT transposases. Based on our biophysical data, we propose guanine tetraplexes as an additional DNA recognition element for binding by RAYT transposase enzymes.

• Klíčová slova
• REP associated tyrosine transposases (RAYTs), bacterial repetitive extragenic palindromes (REP), circular dichroism spectroscopy, interstrand guanine tetraplex, landscape of RAYT DNA recognition elements,
• MeSH
• DNA bakterií chemie   metabolismus   MeSH
• jednovláknová DNA chemie   metabolismus   MeSH
• obrácené repetice genetika   MeSH
• transposasy chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA bakterií MeSH
• jednovláknová DNA MeSH
• transposasy MeSH

We performed PCR of many DNA fragments of 6-32 nucleotides in length. Some of the fragments expanded into kilobase lengths even in the absence of the complementary strand. The dramatic expansion was observed for (CA)8, (TG)8, (CA)4, (CA)6, (CA)12, (TG)4, (TG)6, (TG)12, (TC)10, (GA)10 and other single strands. Similar expansions were exhibited by related trinucleotide repeats (TTG)5, (CAA)5, (TGG)5, and (CCA)5 as well. However even small perturbations of the strict repetitive nature of the DNA primary structure substantially reduced the expansions. The expansion products had properties characteristic for normal Watson-Crick duplexes. Hence either the Taq polymerase and/or other components of the PCR buffer promote homoduplex formation of the nonselfcomplementary fragments, which is necessary to prime the synthesis of the complementary DNA strand, or the Taq polymerase is able to copy the single-stranded DNA template without any priming effect. The present observations have implications for the evolution of genomic DNA, microsatellite length polymorphism as well as the pathological expansions of trinucleotide repeats in the human genome.

• MeSH
• dinukleotidové repetice genetika   MeSH
• expanze repetic DNA genetika   MeSH
• hybridizace nukleových kyselin MeSH
• jednovláknová DNA chemie   genetika   MeSH
• lidé MeSH
• mutageneze genetika   MeSH
• párování bází MeSH
• polymerázová řetězová reakce * MeSH
• sekvence nukleotidů MeSH
• teplota MeSH
• trinukleotidové repetice genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• jednovláknová DNA MeSH