Length Of Single-stranded DNA Research Articles

Nitrogen-doped carbon dots aid in the separation of ssDNA molecules of different length by capillary transient isotachophoresis (ctITP) with laser-induced fluorescence (LIF) detection

This study demonstrates a novel application of nitrogen-doped carbon dots (NCDs) to enable the separation of different lengths of single-stranded DNA (ssDNA) by eletrokinetic means. Carbon dots have recently found widespread application in the fields of sensing, diagnostics, and healthcare due to their biocompatibility and low toxicity. In light of growing interest in the use of ssDNA aptamers over antibodies in the fields of biosensor development and drug delivery, it is important to establish a simple and effective method for aptamer separation. In this study, we employed NCDs as buffer additives in a capillary electrophoresis (CE)-based method, giving rise to the separation of FAM-labeled ssDNA samples ranging from 32 to 100 bases in length, with resolutions ranging from 1.30 – 1.77. In particular, we adopted a capillary transient isotachophoresis (ctITP) system with laser-induced fluorescence (LIF) detection, with both the separation and sample buffers modified by the addition of 30 μg/mL NCDs. These nanomaterials were prepared by a simple hydrothermal method from a mixture of citric acid and ethylenediamine. The NCDs themselves are highly fluorescent and photostable. As components in the background electrolyte, they did not interfere with the fluorescence emission of the FAM-labeled DNA samples. Under the conditions employed, no separation could be achieved in the absence of the NCDs nor with undoped CDs. The results show that NCDs function as buffer additives capable of enhancing electrokinetic-based separations of ssDNA, and hence, provide a new application for these carbon nanomaterials.

An insight into the dependence of the deamination rate of human APOBEC3F on the length of single-stranded DNA, which is affected by the concentrations of APOBEC3F and single-stranded DNA

BackgroundAPOBEC3F (A3F), a member of the human APOBEC3 (A3) family of cytidine deaminases, acts as an anti-HIV-1 factor by deaminating deoxycytidine in the complementary DNA of the viral genome. A full understanding of the deamination behavior of A3F awaits further investigation. MethodsThe real-time NMR method and uracil-DNA glycosylase assay were used to track the activities of the C-terminal domain (CTD) of A3F at different concentrations of A3F-CTD and ssDNA. The steady-state fluorescence anisotropy measurement was used to examine the binding between A3F-CTD and ssDNA with different lengths. The use of the A3F-CTD N214H mutant, having higher activity than the wild-type, facilitated the tracking of the reactions. ResultsA3F-CTD was found to efficiently deaminate the target deoxycytidine in long ssDNA in lower ssDNA concentration conditions ([A3F-CTD] ≫ [ssDNA]), while the target deoxycytidine in short ssDNA is deaminated efficiently in higher ssDNA concentration conditions ([A3F-CTD] ≪ [ssDNA]). This property is quite different from that of the previously studied A3 family member, A3B; the concentrations of the proteins and ssDNA had no effect. ConclusionsThe concentrations of A3F-CTD and ssDNA substrates affect the ssDNA-length-dependence of deamination rate of the A3F-CTD. This unique property of A3F is rationally interpreted on the basis of its binding characteristics with ssDNA. General significanceThe discovery of the unique property of A3F regarding the deamination rate deepens the understanding of its counteraction against HIV-1. Our strategy is applicable to investigate the other aspects of the A3 activities, such as those involved in the cancer development.

Saccharomyces cerevisiae Hrq1 helicase activity is affected by the sequence but not the length of single-stranded DNA

Mutations in the human RecQ4 DNA helicase are associated with three different diseases characterized by genomic instability. To gain insight into how RecQ4 dysfunction leads to these pathologies, several groups have used the Saccharomyces cerevisiae RecQ4 homolog Hrq1 as an experimental model. Hrq1 displays many of the same functions as RecQ4 in vivo and in vitro. However, there is some disagreement in the literature about the effects of single-stranded DNA (ssDNA) length on Hrq1 helicase activity and the ability of Hrq1 to anneal complementary ssDNA oligonucleotides into duplex DNA. Here, we present a side-by-side comparison of Hrq1 and RecQ4 helicase activity, demonstrating that in both cases, long random-sequence 3′ ssDNA tails inhibit DNA unwinding in vitro in a length-dependent manner. This appears to be due to the formation of secondary structures in the random-sequence ssDNA because Hrq1 preferentially unwound poly(dT)-tailed forks independent of ssDNA length. Further, RecQ4 is capable of ssDNA strand annealing and annealing-dependent strand exchange, but Hrq1 lacks these activities. These results establish the importance of DNA sequence in Hrq1 helicase activity, and the absence of Hrq1 strand annealing activity explains the previously identified discrepancies between S. cerevisiae Hrq1 and human RecQ4.

Rejoining of DNA Double-Strand Breaks as a Function of Overhang Length

The ends of spontaneously occurring double-strand breaks (DSBs) may contain various lengths of single-stranded DNA, blocking lesions, and gaps and flaps generated by end annealing. To investigate the processing of such structures, we developed an assay in which annealed oligonucleotides are ligated onto the ends of a linearized plasmid which is then transformed into Saccharomyces cerevisiae. Reconstitution of a marker occurs only when the oligonucleotides are incorporated and repair is in frame, permitting rapid analysis of complex DSB ends. Here, we created DSBs with compatible overhangs of various lengths and asked which pathways are required for their precise repair. Three mechanisms of rejoining were observed, regardless of overhang polarity: nonhomologous end joining (NHEJ), a Rad52-dependent single-strand annealing-like pathway, and a third mechanism independent of the first two mechanisms. DSBs with overhangs of less than 4 bases were mainly repaired by NHEJ. Repair became less dependent on NHEJ when the overhangs were longer or had a higher GC content. Repair of overhangs greater than 8 nucleotides was as much as 150-fold more efficient, impaired 10-fold by rad52 mutation, and highly accurate. Reducing the microhomology extent between long overhangs reduced their repair dramatically, to less than NHEJ of comparable short overhangs. These data support a model in which annealing energy is a primary determinant of the rejoining efficiency and mechanism.

Demonstration of unidirectional single-stranded DNA translocation by PcrA helicase: measurement of step size and translocation speed.

Using a fluorescent sensor for inorganic phosphate, the kinetics of ATP hydrolysis by PcrA helicase were measured in the presence of saturating concentrations of oligonucleotides of various lengths. There is a rapid phase of inorganic phosphate release that is equivalent to several turnovers of the ATPase, followed by slower steady-state ATP hydrolysis. The magnitude of the rapid phase is governed by the length of single-stranded DNA, while the slow phase is independent of its length. A kinetic model is presented in which the rapid phase is associated with translocation along single-stranded DNA, after the PcrA binds randomly along the DNA. There is a linear relationship between the length of single-stranded DNA and both the duration and amplitude of the rapid phase. These data suggest that the translocation activity occurs at 50 bases/s in unidirectional single-base steps, each requiring the hydrolysis of 1 ATP molecule.

DNA base-pair substitutions detected in double-stranded DNA with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

We demonstrate the utility of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for identifying the presence or absence of single base-pair substitutions in a short section of the cystic fibrosis gene. The analysis was performed in two ways: (i) the molecular weight of known lengths of single-stranded DNA from a section of this gene was determined by mass spectrometry, and (ii) short complementary strands from the identical section of the gene were denatured and the difference in molecular weight of the strands was determined. Oligonucleotides corresponding to the wild type sequence were synthesized. The G551D or the R553X mutations were created by introducing single-base substitutions in this oligonucleotide. We recreated double-stranded DNA by annealing each of these three oligonucleotides to complementary oligonucleotides. The MALDI mass spectrometric technique, operating at a mass resolution approaching 1000, correctly identified five of these six oligonucleotides. The most reliable identification was obtained by measuring the difference in molecular weight of the denatured strands. The difference in molecular weight of the complementary strands comprising the wild type, the G551D and the R553X is 52.2 Da, 83.2 Da and 21.2 Da, respectively. The MALDI analysis shows promise as a rapid way to detect the presence of base-pair substitutions without the need for sequencing.