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.

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.

A high sensitivity wireless mass-loading surface acoustic wave DNA biosensor

2014 ◽  
Vol 28 (07) ◽  
pp. 1450056 ◽  
Author(s):  
Hua-Lin Cai ◽  
Yi Yang ◽  
Yi-Han Zhang ◽  
Chang-Jian Zhou ◽  
Cang-Ran Guo ◽  
...  
Keyword(s):  
Acoustic Wave ◽  
Surface Acoustic Wave ◽  
Dna Sequences ◽  
Biological Treatment ◽  
High Performance ◽  
Processing System ◽  
High Sensitivity ◽  
Treatment Method ◽  
Saw Sensor ◽  
Target Dna

In this paper, a surface acoustic wave (SAW) biosensor with gold delay area on LiNbO 3 substrate detecting DNA sequences is proposed. By well-designed device parameters of the SAW sensor, it achieves a high performance for highly sensitive detection of target DNA. In addition, an effective biological treatment method for DNA immobilization and abundant experimental verification of the sensing effect have made it a reliable device in DNA detection. The loading mass of the probe and target DNA sequences is obtained from the frequency shifts, which are big enough in this work due to an effective biological treatment. The experimental results show that the biosensor has a high sensitivity of 1.2 pg/ml/Hz and high selectivity characteristic is also verified by the few responses of other substances. In combination with wireless transceiver, we develop a wireless receiving and processing system that can directly display the detection results.

Restriction of P-element insertions at the Notch locus of Drosophila melanogaster

1987 ◽  
Vol 7 (4) ◽  
pp. 1545-1548
Author(s):  
M R Kelley ◽  
S Kidd ◽  
R L Berg ◽  
M W Young
Keyword(s):  
Drosophila Melanogaster ◽  
Dna Sequences ◽  
Consensus Sequence ◽  
P Element ◽  
Induced Mutations ◽  
P Elements ◽  
Target Dna ◽  
Complex Locus ◽  
Additional Level ◽  
Derivatives Of

P elements move about the Drosophila melanogaster genome in a nonrandom fashion, preferring some chromosomal targets for insertion over others (J. C. J. Eeken, F. H. Sobels, V. Hyland, and A. P. Schalet, Mutat. Res. 150:261-275, 1985; W. R. Engels, Annu. Rev. Genet. 17:315-344, 1983; M. D. Golubovsky, Y. N. Ivanov, and M. M. Green, Proc. Natl. Acad. Sci. USA 74:2973-2975, 1977; M. J. Simmons and J. K. Lim, Proc. Natl. Acad. Sci. USA 77:6042-6046, 1980). Some of this specificity may be due to recognition of a particular DNA sequence in the target DNA; derivatives of an 8-base-pair consensus sequence are occupied by these transposable elements at many different chromosomal locations (K. O'Hare and G. M. Rubin, Cell 34:25-36, 1983). An additional level of specificity of P-element insertions is described in this paper. Of 14 mutations induced in the complex locus Notch by hybrid dysgenesis, 13 involved P-element insertions at or near the transcription start site of the gene. This clustering was not seen in other transposable element-induced mutations of Notch. DNA sequences homologous to the previously described consensus target for P-element insertion are not preferentially located in this region of the locus. The choice of a chromosomal site for integration appears to be based on more subtle variations in chromosome structure that are probably associated with activation or expression of the target gene.

scholarly journals DNA backbone interactions impact the sequence specificity of DNA sulfur-binding domains: revelations from structural analyses

2020 ◽  
Vol 48 (15) ◽  
pp. 8755-8766 ◽  
Author(s):  
Hao Yu ◽  
Jiayi Li ◽  
Guang Liu ◽  
Gong Zhao ◽  
Yuli Wang ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Sulfur Atom ◽  
Dna Sequences ◽  
Restriction Enzymes ◽  
Sequence Specificity ◽  
Structural Determinants ◽  
Hydrogen Bonding Pattern ◽  
Type Iv ◽  
Phosphate Linkage ◽  
Positively Charged

Abstract The sulfur atom of phosphorothioated DNA (PT-DNA) is coordinated by a surface cavity in the conserved sulfur-binding domain (SBD) of type IV restriction enzymes. However, some SBDs cannot recognize the sulfur atom in some sequence contexts. To illustrate the structural determinants for sequence specificity, we resolved the structure of SBDSpr, from endonuclease SprMcrA, in complex with DNA of GPSGCC, GPSATC and GPSAAC contexts. Structural and computational analyses explained why it binds the above PT-DNAs with an affinity in a decreasing order. The structural analysis of SBDSpr–GPSGCC and SBDSco–GPSGCC, the latter only recognizes DNA of GPSGCC, revealed that a positively charged loop above the sulfur-coordination cavity electrostatically interacts with the neighboring DNA phosphate linkage. The structural analysis indicated that the DNA–protein hydrogen bonding pattern and weak non-bonded interaction played important roles in sequence specificity of SBD protein. Exchanges of the positively-charged amino acid residues with the negatively-charged residues in the loop would enable SBDSco to extend recognization for more PT-DNA sequences, implying that type IV endonucleases can be engineered to recognize PT-DNA in novel target sequences.

One-Carbon Metabolism: Linking Nutritional Biochemistry to Epigenetic Programming of Long-Term Development

2019 ◽  
Vol 7 (1) ◽  
pp. 263-287 ◽  
Author(s):  
Constance E. Clare ◽  
Amey H. Brassington ◽  
Wing Yee Kwong ◽  
Kevin D. Sinclair
Keyword(s):  
Dna Sequences ◽  
Methylation Of Dna ◽  
Epigenetic Effects ◽  
Epigenetic Programming ◽  
Target Dna ◽  
Ethnic Variability ◽  
Associated Proteins ◽  
Epigenetic Gene Regulation ◽  
One Carbon Metabolism

One-carbon (1C) metabolism comprises a series of interlinking metabolic pathways that include the methionine and folate cycles that are central to cellular function, providing 1C units (methyl groups) for the synthesis of DNA, polyamines, amino acids, creatine, and phospholipids. S-adenosylmethionine is a potent aminopropyl and methyl donor within these cycles and serves as the principal substrate for methylation of DNA, associated proteins, and RNA. We propose that 1C metabolism functions as a key biochemical conduit between parental environment and epigenetic regulation of early development and that interindividual and ethnic variability in epigenetic-gene regulation arises because of genetic variants within 1C genes, associated epigenetic regulators, and differentially methylated target DNA sequences. We present evidence to support these propositions, drawing upon studies undertaken in humans and animals. We conclude that future studies should assess the epigenetic effects of cumulative (multigenerational) dietary imbalances contemporaneously in both parents, as this better represents the human experience.

scholarly journals Enhancement of target specificity of CRISPR–Cas12a by using a chimeric DNA–RNA guide

2020 ◽  
Vol 48 (15) ◽  
pp. 8601-8616 ◽  
Author(s):  
Hanseop Kim ◽  
Wi-jae Lee ◽  
Yeounsun Oh ◽  
Seung-Hun Kang ◽  
Junho K Hur ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Genome Engineering ◽  
Target Genes ◽  
Target Sequence ◽  
Energy Potential ◽  
Guide Rna ◽  
Sequence Recognition ◽  
Protospacer Adjacent Motif ◽  
Effective Manner ◽  
Target Dna

Abstract The CRISPR–Cas9 system is widely used for target-specific genome engineering. CRISPR–Cas12a (Cpf1) is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cas12a has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and off-target cleavage issues may become more problematic when Cas12a activity is improved for therapeutic purposes. Therefore, we investigated off-target cleavage by Cas12a and modified the Cas12a (cr)RNA to address the off-target cleavage issue. We developed a CRISPR–Cas12a that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR–Cas12a and SpCas9 nickase effectively work in the intracellular genome is suggested. Chimeric guide-based CRISPR- Cas12a genome editing with reduced off-target cleavage, and the resultant, increased safety has potential for therapeutic applications in incurable diseases caused by genetic mutations.

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.

Sign in / Sign up

Export Citation Format

Share Document