scholarly journals Synthetic Elaboration of Native DNA by RASS (SENDR)

2020 ◽  
Author(s):  
Dillon T. Flood ◽  
Kyle W. Knouse ◽  
Julien C. Vantourout ◽  
Brittany Sanchez ◽  
Emily J. Sturgell ◽  
...  
Keyword(s):  
Small Molecules ◽  
Solid Support ◽  
High Yield ◽  
Organic Media ◽  
Two Stage ◽  
Site Specific ◽  
Reversible Adsorption ◽  
Dna Conformations ◽  
Single Coupling ◽  
Stage Process

The controlled, site-specific ligation of molecules to native DNA remains an unanswered challenge. Herein, we report a simple solution to achieve this ligation through the tactical combination of two recently developed technologies: One for the manipulation of DNA in organic media, and another for the chemoselective labeling of alcohols. Reversible Adsorption of Solid Support (RASS) is employed to immobilize DNA and facilitate its transfer into dry acetonitrile. Subsequent ligation with P(V)-based Ψ reagents takes place in high yield with exquisite selectivity for the exposed 3’ or 5’ alcohols on DNA. This two-stage process, dubbed SENDR for Synthetic Elaboration of Native DNA by RASS, can be applied to a multitude of DNA conformations and sequences with a variety of functionalized Ψ reagents to generate useful constructs. Such entities can address numerous longstanding challenges, including the selective single coupling of DNA to proteins, ASOs, and functional small molecules, and also can allow the synthesis of doubly-labeled congeners for novel probe constructs including ones of potential interest to COVID-19 research. Finally, a prototype for the industrialization of SENDR in a kit format is presented.

Synthetic Elaboration of Native DNA by RASS (SENDR)

2020 ◽  
Author(s):  
Dillon T. Flood ◽  
Kyle W. Knouse ◽  
Julien C. Vantourout ◽  
Brittany Sanchez ◽  
Emily J. Sturgell ◽  
...  
Keyword(s):  
Small Molecules ◽  
Solid Support ◽  
High Yield ◽  
Organic Media ◽  
Two Stage ◽  
Site Specific ◽  
Reversible Adsorption ◽  
Dna Conformations ◽  
Single Coupling ◽  
Stage Process

The controlled, site-specific ligation of molecules to native DNA remains an unanswered challenge. Herein, we report a simple solution to achieve this ligation through the tactical combination of two recently developed technologies: One for the manipulation of DNA in organic media, and another for the chemoselective labeling of alcohols. Reversible Adsorption of Solid Support (RASS) is employed to immobilize DNA and facilitate its transfer into dry acetonitrile. Subsequent ligation with P(V)-based Ψ reagents takes place in high yield with exquisite selectivity for the exposed 3’ or 5’ alcohols on DNA. This two-stage process, dubbed SENDR for Synthetic Elaboration of Native DNA by RASS, can be applied to a multitude of DNA conformations and sequences with a variety of functionalized Ψ reagents to generate useful constructs. Such entities can address numerous longstanding challenges, including the selective single coupling of DNA to proteins, ASOs, and functional small molecules, and also can allow the synthesis of doubly-labeled congeners for novel probe constructs including ones of potential interest to COVID-19 research. Finally, a prototype for the industrialization of SENDR in a kit format is presented.

Systematic Engineering of a Protein Nanocage for High-Yield, Site-Specific Modification

2018 ◽  
Author(s):  
Daniel D. Brauer ◽  
Emily C. Hartman ◽  
Daniel L.V. Bader ◽  
Zoe N. Merz ◽  
Danielle Tullman-Ercek ◽  
...  
Keyword(s):  
Small Molecules ◽  
Protein Modification ◽  
Positive Charge ◽  
Specific Protein ◽  
High Yield ◽  
Site Specific ◽  
Protein Cage ◽  
Protein Carriers ◽  
Site Selective ◽  
Targeted Library

<div> <p>Site-specific protein modification is a widely-used strategy to attach drugs, imaging agents, or other useful small molecules to protein carriers. N-terminal modification is particularly useful as a high-yielding, site-selective modification strategy that can be compatible with a wide array of proteins. However, this modification strategy is incompatible with proteins with buried or sterically-hindered N termini, such as virus-like particles like the well-studied MS2 bacteriophage coat protein. To assess VLPs with improved compatibility with these techniques, we generated a targeted library based on the MS2-derived protein cage with N-terminal proline residues followed by three variable positions. We subjected the library to assembly, heat, and chemical selections, and we identified variants that were modified in high yield with no reduction in thermostability. Positive charge adjacent to the native N terminus is surprisingly beneficial for successful extension, and over 50% of the highest performing variants contained positive charge at this position. Taken together, these studies described nonintuitive design rules governing N-terminal extensions and identified successful extensions with high modification potential.</p> </div>

Systematic Engineering of a Protein Nanocage for High-Yield, Site-Specific Modification

2018 ◽  
Author(s):  
Daniel D. Brauer ◽  
Emily C. Hartman ◽  
Daniel L.V. Bader ◽  
Zoe N. Merz ◽  
Danielle Tullman-Ercek ◽  
...  
Keyword(s):  
Small Molecules ◽  
Protein Modification ◽  
Positive Charge ◽  
Specific Protein ◽  
High Yield ◽  
Site Specific ◽  
Protein Cage ◽  
Protein Carriers ◽  
Site Selective ◽  
Targeted Library

<div> <p>Site-specific protein modification is a widely-used strategy to attach drugs, imaging agents, or other useful small molecules to protein carriers. N-terminal modification is particularly useful as a high-yielding, site-selective modification strategy that can be compatible with a wide array of proteins. However, this modification strategy is incompatible with proteins with buried or sterically-hindered N termini, such as virus-like particles like the well-studied MS2 bacteriophage coat protein. To assess VLPs with improved compatibility with these techniques, we generated a targeted library based on the MS2-derived protein cage with N-terminal proline residues followed by three variable positions. We subjected the library to assembly, heat, and chemical selections, and we identified variants that were modified in high yield with no reduction in thermostability. Positive charge adjacent to the native N terminus is surprisingly beneficial for successful extension, and over 50% of the highest performing variants contained positive charge at this position. Taken together, these studies described nonintuitive design rules governing N-terminal extensions and identified successful extensions with high modification potential.</p> </div>

scholarly journals Two-Stage Biodiesel Synthesis from Used Cooking Oil with a High Acid Value via an Ultrasound-Assisted Method

Energies ◽  
2021 ◽  
Vol 14 (12) ◽  
pp. 3703
Author(s):  
Ming-Chien Hsiao ◽  
Wei-Ting Lin ◽  
Wei-Cheng Chiu ◽  
Shuhn-Shyurng Hou
Keyword(s):  
Acid Value ◽  
Molar Ratio ◽  
Optimal Conditions ◽  
Two Stage ◽  
Cooking Oil ◽  
High Acid ◽  
Used Cooking Oil ◽  
Biodiesel Synthesis ◽  
Stage Process ◽  
Ultrasound Assisted

In this study, ultrasound was used to accelerate two-stage (esterification–transesterification) catalytic synthesis of biodiesel from used cooking oil, which originally had a high acid value (4.35 mg KOH/g). In the first stage, acid-catalyzed esterification reaction conditions were developed with a 9:1 methanol/oil molar ratio, sulfuric acid dosage at 2 wt %, and a reaction temperature of 60 °C. Under ultrasound irradiation for 40 min, the acid value was effectively decreased from 4.35 to 1.67 mg KOH/g, which was decreased to a sufficient level (<2 mg KOH/g) to avoid the saponification problem for the subsequent transesterification reaction. In the following stage, base-catalyzed transesterification reactions were carried out with a 12:1 methanol/oil molar ratio, a sodium hydroxide dosage of 1 wt %, and a reaction temperature of 65 °C. Under ultrasound-assisted transesterification for 40 min, the conversion rate of biodiesel reached 97.05%, which met the requirement of EN 14214 standard, i.e., 96.5% minimum. In order to evaluate and explore the improvement of the ultrasound-assisted two-stage (esterification–transesterification) process in shortening the reaction time, additional two-stage biodiesel synthesis experiments using the traditional mechanical stirring method under the optimal conditions were further carried out in this study. It was found that, under the same optimal conditions, using the ultrasound-assisted two-stage process, the total reaction time was significantly reduced to only 80 min, which was much shorter than the total time required by the conventional method of 140 min. It is worth noting that compared with the traditional method without ultrasound, the intensification of the ultrasound-assisted two-stage process significantly shortened the total time from 140 min to 80 min, which is a reduction of 42.9%. It was concluded that the ultrasound-assisted two-stage (esterification–transesterification) catalytic process is an effective and time-saving method for synthesizing biodiesel from used cooking oil with a high acid value.

scholarly journals Site-specific N-glycosylation analysis of animal cell culture-derived Zika virus proteins

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexander Pralow ◽  
Alexander Nikolay ◽  
Arnaud Leon ◽  
Yvonne Genzel ◽  
Erdmann Rapp ◽  
...  
Keyword(s):  
Zika Virus ◽  
Animal Cell ◽  
Gradient Centrifugation ◽  
Viral Envelope ◽  
Protein Glycosylation ◽  
High Yield ◽  
Structural Protein ◽  
Site Specific ◽  
E Protein ◽  
The Impact

AbstractHere, we present for the first time, a site-specific N-glycosylation analysis of proteins from a Brazilian Zika virus (ZIKV) strain. The virus was propagated with high yield in an embryo-derived stem cell line (EB66, Valneva SE), and concentrated by g-force step-gradient centrifugation. Subsequently, the sample was proteolytically digested with different enzymes, measured via a LC–MS/MS-based workflow, and analyzed in a semi-automated way using the in-house developed glyXtoolMS software. The viral non-structural protein 1 (NS1) was glycosylated exclusively with high-mannose structures on both potential N-glycosylation sites. In case of the viral envelope (E) protein, no specific N-glycans could be identified with this method. Nevertheless, N-glycosylation could be proved by enzymatic de-N-glycosylation with PNGase F, resulting in a strong MS-signal of the former glycopeptide with deamidated asparagine at the potential N-glycosylation site N444. This confirmed that this site of the ZIKV E protein is highly N-glycosylated but with very high micro-heterogeneity. Our study clearly demonstrates the progress made towards site-specific N-glycosylation analysis of viral proteins, i.e. for Brazilian ZIKV. It allows to better characterize viral isolates, and to monitor glycosylation of major antigens. The method established can be applied for detailed studies regarding the impact of protein glycosylation on antigenicity and human pathogenicity of many viruses including influenza virus, HIV and corona virus.

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