scholarly journals Protein modification via alkyne hydrosilylation using a substoichiometric amount of ruthenium(ii) catalyst

2017 ◽  
Vol 8 (5) ◽  
pp. 3871-3878 ◽  
Author(s):  
Terence T.-L. Kwan ◽  
Omar Boutureira ◽  
Elizabeth C. Frye ◽  
Stephen J. Walsh ◽  
Moni K. Gupta ◽  
...  
Keyword(s):  
Protein Modification ◽  
Single Site ◽  
Chemical Modifications ◽  
Site Specific ◽  
Specific Modification ◽  
Substoichiometric Amount ◽  
Alkyne Hydrosilylation

The development of site-specific modification of alkyne-functionalized proteins using dimethylarylsilanes and substoichiometric or low-loading of Ru(ii) catalysts is reported. Furthermore, the resultant gem-vinylsilane can undergo further targeted chemical modifications, highlighting its potential for single-site, dual-modification applications.

scholarly journals Multi-site internal modification of long DNA substrates for single-molecule studies

2016 ◽  
Author(s):  
Armando de la Torre ◽  
Yoori Kim ◽  
Andrew A. Leal ◽  
Ilya J. Finkelstein
Keyword(s):  
Single Molecule ◽  
Dna Sequences ◽  
Nuclear Antigen ◽  
Triplet Repeat ◽  
Chemical Modifications ◽  
Site Specific ◽  
Specific Modification ◽  
Modified Dna ◽  
Single Molecule Studies ◽  
Dna Substrates

AbstractSingle-molecule studies of protein-nucleic acid interactions frequently require site-specific modification of long DNA substrates. DNA isolated from bacteriophage λ (λ-DNA) is a convenient source of high quality long (48.5 kb) DNA. However, introducing specific DNA sequences, tertiary structures, and chemical modifications into λ-DNA remains technically challenging. Most current approaches rely on multi-step ligations with low yields and incomplete products. Here, we describe a molecular toolkit for rapid preparation of modified λ-DNA. A set of PCR cassettes facilitates the introduction of recombinant DNA sequences into λ-DNA with 90-100% yield. Furthermore, various DNA structures and chemical modifications can be inserted at user-defined sites via an improved nicking enzyme-based strategy. As a proof-of-principle, we explore the interactions of Proliferating Cell Nuclear Antigen (PCNA) with modified DNA sequences and structures incorporated within λ-DNA. Our results demonstrate that PCNA can load on both 5’-ssDNA flaps and a 13xCAG triplet repeat. However, PCNA remains trapped on the 13xCAG structure, confirming a proposed mechanism for triplet repeat expansion. Although we focus on λ-DNA, this method is applicable to all long DNA substrates. We anticipate that this molecular toolbox will be broadly useful for both ensemble‐ and single-molecule studies that require site-specific modification of long DNA substrates.

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 Mutagenesis by single site-specific arylamine-DNA adducts

1989 ◽  
Vol 264 (33) ◽  
pp. 20120-20130
Author(s):  
P K Gupta ◽  
D L Johnson ◽  
T M Reid ◽  
M S Lee ◽  
L J Romano ◽  
...  
Keyword(s):  
Dna Adducts ◽  
Single Site ◽  
Site Specific

Site-specific modification of E. coli DNA polymerase I large fragment with pyridoxal 5'-phosphate

Biochemistry ◽  
1984 ◽  
Vol 23 (9) ◽  
pp. 2073-2078 ◽  
Author(s):  
Anup K. Hazra ◽  
Sevilla Detera-Wadleigh ◽  
Samuel H. Wilson
Keyword(s):  
Dna Polymerase ◽  
Large Fragment ◽  
Dna Polymerase I ◽  
Site Specific ◽  
E Coli ◽  
Specific Modification ◽  
Polymerase I

scholarly journals Dual, Site-Specific Modification of Antibodies by Using Solid-Phase Immobilized Microbial Transglutaminase

ChemBioChem ◽  
2017 ◽  
Vol 18 (19) ◽  
pp. 1923-1927 ◽  
Author(s):  
Philipp R. Spycher ◽  
Christian A. Amann ◽  
Jöri E. Wehrmüller ◽  
David R. Hurwitz ◽  
Olivier Kreis ◽  
...  
Keyword(s):  
Solid Phase ◽  
Microbial Transglutaminase ◽  
Site Specific ◽  
Specific Modification ◽  
Dual Site

scholarly journals Expression of ΔF508 CFTR in normal mouse lung after site-specific modification of CFTR sequences by SFHR

Gene Therapy ◽  
2001 ◽  
Vol 8 (12) ◽  
pp. 961-965 ◽  
Author(s):  
KK Goncz ◽  
A Colosimo ◽  
B Dallapiccola ◽  
L Gagné ◽  
K Hong ◽  
...  
Keyword(s):  
Normal Mouse ◽  
Mouse Lung ◽  
Site Specific ◽  
Specific Modification ◽  
Δf508 Cftr

Comparison of the patterns of chlamydia and gonorrhoea at the oropharynx, anorectum and urethra among men who have sex with men

2021 ◽  
pp. sextrans-2020-054632
Author(s):  
Marjan Tabesh ◽  
Christopher K Fairley ◽  
Jane S Hocking ◽  
Deborah A Williamson ◽  
Lei Zhang ◽  
...  
Keyword(s):  
Health Centre ◽  
Single Site ◽  
Chlamydia Infection ◽  
Similar Proportion ◽  
Anatomical Site ◽  
Site Infection ◽  
Site Specific ◽  
Sexually Transmitted ◽  
Sex With Men ◽  
First Time

ObjectiveChlamydia and gonorrhoea are common sexually transmitted infections that infect the oropharynx, anorectum and urethra in men who have sex with men (MSM). This study aimed to examine the pattern of infection at more than one site (multisite) for chlamydia and gonorrhoea among MSM.MethodsThis was a retrospective study of MSM attending the Melbourne Sexual Health Centre for the first time between 2018 and 2019. We included MSM aged ≥16 years who had tested for Neisseria gonorrhoeae and Chlamydia trachomatis at all three sites (oropharynx, anorectum and urethra). We compared infections that occurred at a single site (termed single-site infection) and those that occurred at more than one site (termed multisite infections).ResultsOf the 3938 men who were tested for chlamydia and gonorrhoea, 498/3938 men (12.6%, 95% CI 11.5% to 13.6%) had chlamydia at any site, of whom 400/498 (80.3%, 95% CI 78.9% to 81.2%) had single-site chlamydia infection, and 98/498 (19.7%, 95% CI 16.2% to 23.1%) had multisite infections. A similar proportion of men had gonorrhoea at any site (447/3938, 11.4%, 95% CI 10.3% to 12.2%), but among these 447 men, single-site infection was less common (256/447, 57.3%, 95% CI 52.6% to 61.7%, p<0.001) and multisite infection (191/447, 42.7%, 95% CI 38.2% to 47.3%, p<0.001) was more common than chlamydia. There were also marked differences by anatomical site. Urethral infection commonly occurred as single sites (75/122, 61.5%, 95% CI 52.8% to 70.1%) for chlamydia but uncommonly occurred for gonorrhoea (12/100, 12.0%, 95% CI 5.6% to 18.3%, p<0.001). In contrast, anorectal infection uncommonly occurred as multisite infection for chlamydia (98/394, 24.9%, 95% CI 20.6% to 29.1%) but was common (184/309, 59.5%, 95% CI 54.0% to 64.9%, p<0.001) for gonorrhoea.ConclusionsThe markedly different pattern of site-specific infection for chlamydia and gonorrhoea infections among the same MSM suggests significant differences in the transmissibility between anatomical sites and the duration of each infection at each site.

scholarly journals Site-Specific and Trigger-Activated Modification of Proteins by Means of Catalytic Hemin/G-quadruplex (hGQ) DNAzyme Nanostructures

2020 ◽  
Author(s):  
Jordi Keijzer ◽  
Bauke Albada
Keyword(s):  
Heavy Chain ◽  
Protein Modification ◽  
Site Specificity ◽  
Site Specific ◽  
Antibody Drug Conjugates ◽  
Synthetic Dna ◽  
Drug Conjugates ◽  
Sds Page ◽  
G Quadruplex ◽  
Antibody Drug

<div>Synthetic DNA that forms various G-quadruplex nanostructures, in combination with hemin, <i>N</i>-methyl luminol derivatives, and H2O2 can site-specifically modify proteins (i.e. evidence is provided for lysozyme and human alpha-thrombin). The catalytic modification is completed in 15-30 mins, and the site-specificity is influenced by the G-quadruplex topology (a total of 22 G-quadruplex forming sequences was tested). We also show that the heavy chain of the therapeutic antibody trastuzumab is modified, which facilitates the preparation of antibody-drug conjugates. Furthermore, a trigger can be programmed into this synthetic DNA so that the protein modification chemistry is made dependent on an external trigger.</div><div><br></div>Techniques used: HPLC, SDS-PAGE, LC-MS/MS, NMR.

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