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 Terminal lipophilization of a unique DNA dodecamer by various nucleolipid headgroups: Their incorporation into artificial lipid bilayers and hydrodynamic properties

2015 ◽  
Vol 11 ◽  
pp. 913-929 ◽  
Author(s):  
Emma Werz ◽  
Helmut Rosemeyer
Keyword(s):  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Single Molecule ◽  
Head Group ◽  
Artificial Lipid Bilayers ◽  
Decisive Importance ◽  
Head Groups ◽  
Single Molecule Fluorescence Spectroscopy ◽  
Dna Dodecamer ◽  
And Diffusion

A series of six cyanine-5-labeled oligonucleotides (LONs 10–15), each terminally lipophilized with different nucleolipid head groups, were synthesized using the recently prepared phosphoramidites 4b–9b. The insertion of the LONs within an artificial lipid bilayer, composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), was studied by single molecule fluorescence spectroscopy and microscopy with the help of an optically transparent microfluidic sample carrier with perfusion capabilities. The incorporation of the lipo-oligonucleotides into the bilayer was studied with respect to efficiency (maximal bilayer brightness) as well as stability against perfusion (final stable bilayer brightness). Attempts to correlate these parameters with the log P values of the corresponding nucleolipid head groups failed, a result which clearly demonstrates that not only the lipophilicity but mainly the chemical structure and topology of the head group is of decisive importance for the optimal interaction of a lipo-oligonucleotide with an artificial lipid bilayer. Moreover, fluorescence half-live and diffusion time values were measured to determine the diffusion coefficients of the lipo-oligonucleotides.

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 Real-Time Detection of Genetically Modified Soya Using Lightcycler and ABI 7700 Platforms with TaqMan, Scorpion, and SYBR Green I Chemistries

2002 ◽  
Vol 85 (4) ◽  
pp. 938-944 ◽  
Author(s):  
Catherine F Terry ◽  
Della J Shanahan ◽  
Lydia D Ballam ◽  
Neil Harris ◽  
David G McDowell ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Sequences ◽  
Genetically Modified Organisms ◽  
Genetically Modified ◽  
Sybr Green ◽  
Sybr Green I ◽  
Quantitative Detection ◽  
Roundup Ready ◽  
Detection Systems ◽  
Copy Numbers

Abstract A comparative cross platform evaluation of real-time polymerase chain reaction detection of DNA sequences present in Roundup Ready soya was undertaken using the ABI 7700 and Roche Lightcycler detection systems in combination with 3 different detection chemistries: TaqMan, Scorpion primers, and SYBR Green I fluorescent dye. Various copy numbers of a plasmid containing the soya lectin sequence were used to determine the sensitivity and reproducibility of the different technology combinations and to examine both inter and intra machine variability. To examine the relative accuracy of each technology, the genetically modified soya content of baked products containing known amounts of Roundup Ready soya was determined by detection of lectin and the EPSPS transgene. It was determined that the combination of TaqMan detection chemistry and the ABI 7700 platform represented the best method for quantitative detection of genetically modified organisms in terms of both precision and accuracy.

Experimental Validation of DNA Sequences for DNA Computing: Use of a SYBR Green I Assay

2006 ◽  
pp. 248-256 ◽  
Author(s):  
Wendy K. Pogozelski ◽  
Matthew P. Bernard ◽  
Salvatore F. Priore ◽  
Anthony J. Macula
Keyword(s):  
Dna Sequences ◽  
Dna Computing ◽  
Experimental Validation ◽  
Sybr Green ◽  
Sybr Green I

The influence of SYBR Green I on isothermal amplification with Bst exo DNA polymerase.

Biomics ◽  
2018 ◽  
Vol 10 (3) ◽  
pp. 268-273
Author(s):  
A.R. Gilvanov ◽  
A.R. Sakhabutdinova ◽  
A.V. Chemeris ◽  
R.R. Garafutdinov
Keyword(s):  
Dna Polymerase ◽  
Sybr Green ◽  
Isothermal Amplification ◽  
Sybr Green I
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