scholarly journals Preparation and Application of Triple Helix Forming Oligonucleotides and Single Strand Oligonucleotide Donors for Gene Correction

2014 ◽  
pp. 103-113 ◽  
Author(s):  
Md. Rowshon Alam ◽  
Arun Kalliat Thazhathveetil ◽  
Hong Li ◽  
Michael M. Seidman
Keyword(s):  
Triple Helix ◽  
Single Strand ◽  
Gene Correction

scholarly journals Relative Stability of the Scleroglucan Triple-Helix and Single Strand: an Insight from Computational and Experimental Techniques

2016 ◽  
Vol 230 (9) ◽  
Author(s):  
Gianfranco Bocchinfuso ◽  
Claudia Mazzuca ◽  
Paolo Conflitti ◽  
Davide Cori ◽  
Tommasina Coviello ◽  
...  
Keyword(s):  
Relative Stability ◽  
Triple Helix ◽  
Hydrophobic Interactions ◽  
Single Strand ◽  
Single Chain ◽  
Ph Values ◽  
Partial Unfolding ◽  
Triplex Structure ◽  
Partially Unfolded ◽  
The Stability

AbstractScleroglucan (Sclg) is a polysaccharide that exhibits a triple helix conformation (triplex), both in aqueous solution and in the solid state, which is lost in DMSO solution, at high temperature and at high pH values. The triplex conformation is characterized by a high rigidity, responsible of Sclg peculiar properties. Although the relative stability of triplex and single strand has already been investigated, different structural details are still missing.In the present study, we analyse the structural properties and the factors stabilizing the single chain and the triple helix of Sclg in different conditions. To this end, we simulated both systems in water and in DMSO. The triple helix has been also simulated in the presence of chemical damages on one of the three strands (to reproduce in silico the effect of sonication) or by inducing a partial unfolding of the triplex structure. The computational results have been compared with experimental evidences in which the triplex denaturation, at alkaline pH values, has been followed by monitoring the UV and CD spectra of Congo red, used as a probe molecule. Our results indicate that sonication breaks the Sclg chains without appreciably changing the stability of the other tracts of triple helix. The simulated perturbed or partially unfolded triplexes show a clear tendency to form less ordered aggregates. Finally, our simulations put in evidence an important role of the hydrophobic interactions both in the triplex stability and in the aggregation processes observed after induced denaturation.

scholarly journals Priming DNA Replication from Triple Helix Oligonucleotides: Possible Threestranded DNA in DNA Polymerases

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Patrick P. Lestienne
Keyword(s):  
Dna Replication ◽  
Triple Helix ◽  
Dna Polymerases ◽  
Direct Repeat ◽  
Single Strand ◽  
Homologous Sequence ◽  
Duplex Dna ◽  
Dna Rearrangements ◽  
Initiation Of Replication ◽  
Strand Breakage

Triplex associate with a duplex DNA presenting the same polypurine or polypyrimidine-rich sequence in an antiparallel orientation. So far, triplex forming oligonucleotides (TFOs) are known to inhibit transcription, replication, and to induce mutations. A new property of TFO is reviewed here upon analysis of DNA breakpoint yielding DNA rearrangements; the synthesized sequence of the first direct repeat displays a skewed polypurine- rich sequence. This synthesized sequence can bind the second homologous duplex sequence through the formation of a triple helix, which is able to prime further DNA replication. In these case, the d(G)-rich Triple Helix Primers (THP) bind the homologous strand in a parallel manner, possibly via a RecA-like mechanism. This novel property is shared by all tested DNA polymerases: phage, retrovirus, bacteria, and human. These features may account for illegitimate initiation of replication upon single-strand breakage and annealing to a homologous sequence where priming may occur. Our experiments suggest that DNA polymerases can bind three instead of two polynucleotide strands in their catalytic centre.

scholarly journals Efficient gene correction of an aberrant splice site in β‐thalassaemia iPSCs by CRISPR/Cas9 and single‐strand oligodeoxynucleotides

2019 ◽  
Vol 23 (12) ◽  
pp. 8046-8057 ◽  
Author(s):  
Zeyu Xiong ◽  
Yingjun Xie ◽  
Yi Yang ◽  
Yanting Xue ◽  
Ding Wang ◽  
...  
Keyword(s):  
Splice Site ◽  
Single Strand ◽  
Aberrant Splice ◽  
Gene Correction ◽  
Efficient Gene

scholarly journals Development of a Clinically Applicable Method for High-Efficiency Gene Correction of Plerixafor-Mobilized CD34+ Cells from Patients with Sickle Cell Disease

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2191-2191 ◽  
Author(s):  
Naoya Uchida ◽  
Linhong Li ◽  
Juan J Haro-Mora ◽  
Selami Demirci ◽  
Tina Nassehi ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Globin Gene ◽  
Erythroid Differentiation ◽  
Single Strand ◽  
Gene Correction ◽  
Guide Rna ◽  
Protein Levels ◽  
Cd34 Cells ◽  
Cas9 Mrna

Abstract Sickle cell disease (SCD) is caused by a 20A>T mutation in the β-globin gene, and can be cured by therapeutic β-globin gene addition into hematopoietic stem cells (HSCs) with lentiviral transduction. However, this method relies upon random integration, leaving the SCD mutation intact and potentially inducing insertional mutagenesis. Genome editing technologies have the potential to correct the SCD mutation without integration, producing adult hemoglobin (Hb) while simultaneously eliminating sickle Hb. In this study, we investigated CRISPR/Cas9-based gene correction for SCD CD34+ cells. Plerixafor-mobilized SCD CD34+ cells were transfected by electroporation using the GMP-compliant, FDA Master File-supported, and scalable MaxCyte GT System to deliver SCD mutation-specific guide RNA at 200mg/ml, SpCas9 mRNA at 200mg/ml or protein at 120mg/ml, and single strand donor DNA with a normal β-globin sequence at 80, 120, or 200mg/ml. We chose Cas9 mRNA and single strand donor DNA due to the ease of clinical grade large-scale production and to avoid the need for viral vector manufacturing. Following erythroid differentiation, gene correction efficiency was evaluated at DNA levels by deep sequencing and at protein levels by reverse-phase HPLC. Cell viability was reduced to 76-87% after electroporation, compared to 90% in the control. We observed high-efficiency genome editing (29-34% gene correction and 49-58% indels) with Cas9 mRNA, showing donor DNA concentration dependence, and editing levels were comparable to Cas9 protein (39% correction and 43% indels). 15-23% Biallelic and 17-26% monoallelic gene correction were detected at the clonal level by colony assay. After erythroid differentiation, up to 54% normal β-globin production was observed with Cas9 mRNA (Figure), comparable to Cas9 protein (67%), while βs-globin amounts were markedly reduced under both conditions (6-10%). Similar correction efficiencies were obtained from two additional SCD patients' CD34+ cells at DNA levels (28-35%) and protein levels (33-56%). These data demonstrate that Cas9 mRNA and single strand donor DNA allow for efficient gene correction in SCD CD34+ cells, exceeding the therapeutic threshold of 20% in SCD. We then evaluated off-target effects on the δ-globin gene, which was reported as a major off-target site in β-globin gene editing due to high homology; however, almost no off-target effects (0.6-1.3% indels) were detected. Interestingly, gene conversion in the 9T>C polymorphism (11bp upstream of SCD mutation) on the β-globin gene was observed, and this conversion always occurred with SCD gene correction (26-33% of SCD gene correction), suggesting that gene conversion is strongly affected by distance from the target site. In addition, we evaluated genome editing among subpopulations of CD34+ cells from 3 healthy donors under the same conditions (normal β-globin to SCD mutation). We observed similar editing efficiencies (conversion and indels) among more immature (CD34+CD133+CD90+) and relatively differentiated populations (CD34+CD133+CD90-, CD34+CD133-, and CD34-) as well as among cells at different phases of the cell cycle (G0/G1, S, and G2/M), suggesting that similar gene correction efficiencies are obtained in all CD34+ cell populations, including the HSC population. We have begun efforts to evaluate gene-corrected SCD CD34+ cell engraftment in the mouse xenograft model, as similarly corrected X-CGD CD34+ cells were engrafted in immunodeficient mice. To examine the effects of indels in the β-globin gene, we next evaluated Hb production from genome-edited SCD CD34+ cells (2 patients) without donor DNA. Editing without donor DNA resulted in 63-70% indels (compared to 26-29% correction and 46-53% indels with donor DNA) and increased non-adult Hb production (small amounts of fetal Hb and significant amounts of a Hb variant), which will require further investigation to characterize. In summary, we observed efficient gene correction in SCD CD34+ cells with a simple Cas9 mRNA, single strand donor DNA, and guide RNA method, resulting in ~30% gene correction and ~50% indels. After erythroid differentiation, the majority of Hb detected was adult Hb; we detected up to 54% normal β-globin production with a marked reduction of βs-globin to ~10%. Evaluation of engraftment potential is required for gene-corrected CD34+ cells, but these methods would be clinically applicable for gene correction in SCD. Figure. Figure. Disclosures Li: MaxCyte, Inc.: Employment. Allen:MaxCyte, Inc.: Employment. Peshwa:MaxCyte, Inc.: Employment.

Single-strand DNA triple-helix formation

Biochemistry ◽  
1990 ◽  
Vol 29 (42) ◽  
pp. 9761-9765 ◽  
Author(s):  
Robert Haner ◽  
Peter B. Dervan
Keyword(s):  
Triple Helix ◽  
Single Strand ◽  
Helix Formation ◽  
Triple Helix Formation ◽  
Dna Triple Helix

“Triple Helix” in Russia’s Innovation System

2007 ◽  
pp. 123-135 ◽  
Author(s):  
I. Dezhina ◽  
V. Kiseleva
Keyword(s):  
Triple Helix ◽  
Government Regulation ◽  
Innovation System ◽  
Innovation Systems ◽  
Modern Theory

The article analyzes modern theory that explains the specificity of relationships among government, science and business in innovation systems - the "triple helix" concept. Factors that determine the appearance of new theory are systematized. The peculiarities of formation of "triple helix" in Russia are described, including the development of science, business and the system of government regulation in innovation sphere. The conclusion is made that currently in Russia only double linkages are formed.

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