scholarly journals How abasic sites impact hole transfer dynamics in GC-rich DNA sequences

2018 ◽  
Vol 20 (35) ◽  
pp. 23123-23131
Author(s):  
Marina Corbella ◽  
Alexander A. Voityuk ◽  
Carles Curutchet
Keyword(s):  
Dna Sequences ◽  
Dna Duplexes ◽  
Hole Transfer ◽  
Abasic Sites

Hole transfer dynamics through GC-rich DNA duplexes containing abasic sites is strongly modulated by the nature of the unpaired nucleobase.

scholarly journals Specific DNA duplex formation at an artificial lipid bilayer: fluorescence microscopy after Sybr Green I staining

2014 ◽  
Vol 10 ◽  
pp. 2307-2321 ◽  
Author(s):  
Emma Werz ◽  
Helmut Rosemeyer
Keyword(s):  
Lipid Bilayer ◽  
Single Molecule ◽  
Dna Sequences ◽  
Sybr Green ◽  
Sybr Green I ◽  
Racemic Mixture ◽  
Dna Duplexes ◽  
Target Dna ◽  
Single Molecule Fluorescence Spectroscopy ◽  
Duplex Formation

The article describes the immobilization of different probe oligonucleotides (4, 7, 10) carrying each a racemic mixture of 2,3-bis(hexadecyloxy)propan-1-ol (1a) at the 5’-terminus on a stable artificial lipid bilayer composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The bilayer separates two compartments (cis/trans channel) of an optical transparent microfluidic sample carrier with perfusion capabilities. Injection of unlabeled target DNA sequences (6, 8, or 9), differing in sequence and length, leads in the case of complementarity to the formation of stable DNA duplexes at the bilayer surface. This could be verified by Sybr Green I double strand staining, followed by incubation periods and thorough perfusions, and was visualized by single molecule fluorescence spectroscopy and microscopy. The different bilayer-immobilized complexes consisting of various DNA duplexes and the fluorescent dye were studied with respect to the kinetics of their formation as well as to their stability against perfusion.

scholarly journals Nonreciprocal exchanges of information between DNA duplexes coinjected into mammalian cell nuclei.

1985 ◽  
Vol 5 (1) ◽  
pp. 59-69 ◽  
Author(s):  
K R Folger ◽  
K Thomas ◽  
M R Capecchi
Keyword(s):  
Homologous Recombination ◽  
Dna Sequences ◽  
Plasmid Dna ◽  
Mammalian Cells ◽  
Recombinant Dna ◽  
Dna Duplexes ◽  
Dna Molecules ◽  
Cell Nuclei ◽  
High Concentration ◽  
Length Polymorphisms

We have examined the mechanism of homologous recombination between plasmid molecules coinjected into cultured mammalian cells. Cell lines containing recombinant DNA molecules were obtained by selecting for the reconstruction of a functional Neor gene from two plasmids that bear different amber mutations in the Neor gene. In addition, these plasmids contain restriction-length polymorphisms within and near the Neor gene. These polymorphisms did not confer a selectable phenotype but were used to identify and categorize selected and nonselected recombinant DNA molecules. The striking conclusion from this analysis is that the predominant mechanism for the exchange of information between coinjected plasmid molecules over short distances (i.e., less than 1 kilobase) proceeds via nonreciprocal homologous recombination. The frequency of homologous recombination between coinjected plasmid molecules in cultured mammalian cells is extremely high, approaching unity. We demonstrate that this high frequency requires neither a high input of plasmid molecules per cell nor a localized high concentration of plasmid DNA within the nucleus. Thus, it appears that plasmid molecules, once introduced into the nucleus, have no difficulty seeking each other out and participating in homologous recombination even in the presence of a vast excess of host DNA sequences. Finally, we show that most of the homologous recombination events occur within a 1-h interval after the introduction of plasmid DNA into the cell nucleus.

scholarly journals Rapid assessment of Watson–Crick to Hoogsteen exchange in unlabeled DNA duplexes using high-power SELOPE imino <sup>1</sup>H CEST

2021 ◽  
Vol 2 (2) ◽  
pp. 715-731
Author(s):  
Bei Liu ◽  
Atul Rangadurai ◽  
Honglue Shi ◽  
Hashim M. Al-Hashimi
Keyword(s):  
High Power ◽  
Dna Sequences ◽  
Dynamic Equilibrium ◽  
Chemical Exchange ◽  
Rapid Assessment ◽  
Relaxation Dispersion ◽  
Duplex Dna ◽  
Dna Duplexes ◽  
Base Pairs ◽  
1H Cest

Abstract. In duplex DNA, Watson–Crick A–T and G–C base pairs (bp's) exist in dynamic equilibrium with an alternative Hoogsteen conformation, which is low in abundance and short-lived. Measuring how the Hoogsteen dynamics varies across different DNA sequences, structural contexts and physiological conditions is key for identifying potential Hoogsteen hot spots and for understanding the potential roles of Hoogsteen base pairs in DNA recognition and repair. However, such studies are hampered by the need to prepare 13C or 15N isotopically enriched DNA samples for NMR relaxation dispersion (RD) experiments. Here, using SELective Optimized Proton Experiments (SELOPE) 1H CEST experiments employing high-power radiofrequency fields (B1 > 250 Hz) targeting imino protons, we demonstrate accurate and robust characterization of Watson–Crick to Hoogsteen exchange, without the need for isotopic enrichment of the DNA. For 13 residues in three DNA duplexes under different temperature and pH conditions, the exchange parameters deduced from high-power imino 1H CEST were in very good agreement with counterparts measured using off-resonance 13C / 15N spin relaxation in the rotating frame (R1ρ). It is shown that 1H–1H NOE effects which typically introduce artifacts in 1H-based measurements of chemical exchange can be effectively suppressed by selective excitation, provided that the relaxation delay is short (≤ 100 ms). The 1H CEST experiment can be performed with ∼ 10× higher throughput and ∼ 100× lower cost relative to 13C / 15N R1ρ and enabled Hoogsteen chemical exchange measurements undetectable by R1ρ. The results reveal an increased propensity to form Hoogsteen bp's near terminal ends and a diminished propensity within A-tract motifs. The 1H CEST experiment provides a basis for rapidly screening Hoogsteen breathing in duplex DNA, enabling identification of unusual motifs for more in-depth characterization.

scholarly journals DNA polymerase activity on synthetic N3′→P5′ phosphoramidate DNA templates

2019 ◽  
Vol 47 (17) ◽  
pp. 8941-8949 ◽  
Author(s):  
Victor S Lelyveld ◽  
Derek K O’Flaherty ◽  
Lijun Zhou ◽  
Enver Cagri Izgu ◽  
Jack W Szostak
Keyword(s):  
Dna Polymerase ◽  
Dna Sequences ◽  
Chemical Space ◽  
Genetic Material ◽  
Polymerase Activity ◽  
Dna Duplexes ◽  
Dna Templates ◽  
Reverse Transcriptases ◽  
The Universe ◽  
Rna And Dna

Abstract Genetic polymers that could plausibly govern life in the universe might inhabit a broad swath of chemical space. A subset of these genetic systems can exchange information with RNA and DNA and could therefore form the basis for model protocells in the laboratory. N3′→P5′ phosphoramidate (NP) DNA is defined by a conservative linkage substitution and has shown promise as a protocellular genetic material, but much remains unknown about its functionality and fidelity due to limited enzymatic tools. Conveniently, we find widespread NP-DNA-dependent DNA polymerase activity among reverse transcriptases, an observation consistent with structural studies of the RNA-like conformation of NP-DNA duplexes. Here, we analyze the consequences of this unnatural template linkage on the kinetics and fidelity of DNA polymerization activity catalyzed by wild-type and variant reverse transcriptases. Template-associated deficits in kinetics and fidelity suggest that even highly conservative template modifications give rise to error-prone DNA polymerase activity. Enzymatic copying of NP-DNA sequences is nevertheless an important step toward the future study and engineering of this synthetic genetic polymer.

Fast, inexpensive, high-yielding, site-selective, chemical synthesis of cross-linked DNA duplexes via hydrazone formation between N[superscript 4]-aminocytidine and Ap-sites

2017 ◽  
Author(s):  
◽  
Jacqueline Gamboa Varela
Keyword(s):  
High Yield ◽  
Dna Duplexes ◽  
Cross Link ◽  
University Of Missouri ◽  
Cellular Processes ◽  
Abasic Sites ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
The Cross ◽  
High Yields

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] DNA is the central molecule of biology as it stores the genetic information for cells to properly function and develop. Modifications to the DNA can stall cellular processes such as replication and transcription, leading the cell to recruit repair machinery or in some cases undergo apoptosis. Interstrand cross-links are particularly significant types of DNA damage because they prevent strand separation required for replication and transcription. Cross-links involve bonding between the two strands of DNA. The rate and mechanism of cross-link repair in cells are not well understood. A significant challenge in the study of cross-link repair is the synthesis of chemically well-defined DNA cross-links. Here we summarize the preparation of cross-links derived from the hydrazone formation between a non-natural nucleobase N4-aminocytidine and abasic sites in duplex DNA. The cross-link was generated rapidly and in high yield. The cross-link is stable under physiological conditions but, interestingly, can be reversibly dissociated and re-formed by thermal cycling between 20-80 [degrees]C. We provided evidence that the cross-link is stable against multiple agents and the cross-link is reversible. We used this chemistry to prepare structurally diverse cross-links for the utilization in cross-link repair studies. Overall, we developed a synthetic cross-link that is easily and rapidly prepared from commercially available reagents in high yields, at defined locations in duplexed DNA.

Surface-enhanced Raman spectroscopy (SERS) characterisation of abasic sites in DNA duplexes

The Analyst ◽  
2019 ◽  
Vol 144 (23) ◽  
pp. 6862-6865
Author(s):  
Luca Guerrini ◽  
Ramon A. Alvarez-Puebla
Keyword(s):  
Raman Spectroscopy ◽  
Surface Enhanced Raman Spectroscopy ◽  
Dna Duplexes ◽  
Structural Characterisation ◽  
P Acquisition ◽  
Abasic Sites ◽  
Surface Enhanced ◽  
Surface Enhanced Raman

Acquisition of the intrinsic SERS spectra of abasic sites containing DNA enables their structural characterisation and discrimination.

scholarly journals Structural Determinants of Photoreactivity of Triplex Forming Oligonucleotides Conjugated to Psoralens

2010 ◽  
Vol 2010 ◽  
pp. 1-7 ◽  
Author(s):  
Rajagopal Krishnan ◽  
Dennis H. Oh
Keyword(s):  
Dna Damage ◽  
Optimal Design ◽  
Dna Sequences ◽  
Dna Duplexes ◽  
Cd Spectra ◽  
Structural Determinants ◽  
Binding Ability ◽  
Target Dna ◽  
Rna Backbone ◽  
Functional Consequences

Triplex-forming oligonucleotides (TFOs) with both DNA and2′-O-methyl RNA backbones can direct psoralen photoadducts to specific DNA sequences. However, the functional consequences of these differing structures on psoralen photoreactivity are unknown. We designed TFO sequences with DNA and2′-O-methyl RNA backbones conjugated to psoralen by 2-carbon linkers and examined their ability to bind and target damage to model DNA duplexes corresponding to sequences within the humanHPRTgene. While TFO binding affinity was not dramatically affected by the type of backbone, psoralen photoreactivity was completely abrogated by the2′-O-methyl RNA backbone. Photoreactivity was restored when the psoralen was conjugated to the RNA TFO via a 6-carbon linker. In contrast to the B-form DNA of triplexes formed by DNA TFOs, the CD spectra of triplexes formed with2′-O-methyl RNA TFOs exhibited features of A-form DNA. These results indicate that2′-O-methyl RNA TFOs induce a partial B-to-A transition in their target DNA sequences which may impair the photoreactivity of a conjugated psoralen and suggest that optimal design of TFOs to target DNA damage may require a balance between binding ability and drug reactivity.

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