scholarly journals Photocrosslinking of cDNA Display Molecules with Their Target Proteins as a New Strategy for Peptide Selection

Molecules ◽  
2020 ◽  
Vol 25 (6) ◽  
pp. 1472
Author(s):  
Takuya Terai ◽  
Tomoyuki Koike ◽  
Naoto Nemoto
Keyword(s):  
Uv Light ◽  
Specific Binding ◽  
Light Irradiation ◽  
Library Size ◽  
Peptide Aptamers ◽  
Target Proteins ◽  
New Strategy ◽  
Straightforward Solution ◽  
Selection Of

Binding peptides for given target molecules are often selected in vitro during drug discovery and chemical biology research. Among several display technologies for this purpose, complementary DNA (cDNA) display (a covalent complex of a peptide and its encoding cDNA linked via a specially designed puromycin-conjugated DNA) is unique in terms of library size, chemical stability, and flexibility of modification. However, selection of cDNA display libraries often suffers from false positives derived from non-specific binding. Although rigorous washing is a straightforward solution, this also leads to the loss of specific binders with moderate affinity because the interaction is non-covalent. To address this issue, herein, we propose a method to covalently link cDNA display molecules with their target proteins using light irradiation. We designed a new puromycin DNA linker that contains a photocrosslinking nucleic acid and prepared cDNA display molecules using the linker. Target proteins were also labeled with a short single-stranded DNA that should transiently hybridize with the linker. Upon ultraviolet (UV) light irradiation, cDNA display molecules encoding correct peptide aptamers made stable crosslinked products with the target proteins in solution, while display molecules encoding control peptides did not. Although further optimization and improvement is necessary, the results pave the way for efficient selection of peptide aptamers in multimolecular crowding biosystems.

scholarly journals In vitro and in vivo inhibition of malaria parasite infection by monoclonal antibodies against Plasmodium falciparum circumsporozoite protein (CSP)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Merricka C. Livingstone ◽  
Alexis A. Bitzer ◽  
Alish Giri ◽  
Kun Luo ◽  
Rajeshwer S. Sankhala ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Monoclonal Antibodies ◽  
Disease Burden ◽  
Vaccine Candidate ◽  
Specific Binding ◽  
Circumsporozoite Protein ◽  
Target Epitope ◽  
Selection Of

AbstractPlasmodium falciparum malaria contributes to a significant global disease burden. Circumsporozoite protein (CSP), the most abundant sporozoite stage antigen, is a prime vaccine candidate. Inhibitory monoclonal antibodies (mAbs) against CSP map to either a short junctional sequence or the central (NPNA)n repeat region. We compared in vitro and in vivo activities of six CSP-specific mAbs derived from human recipients of a recombinant CSP vaccine RTS,S/AS01 (mAbs 317 and 311); an irradiated whole sporozoite vaccine PfSPZ (mAbs CIS43 and MGG4); or individuals exposed to malaria (mAbs 580 and 663). RTS,S mAb 317 that specifically binds the (NPNA)n epitope, had the highest affinity and it elicited the best sterile protection in mice. The most potent inhibitor of sporozoite invasion in vitro was mAb CIS43 which shows dual-specific binding to the junctional sequence and (NPNA)n. In vivo mouse protection was associated with the mAb reactivity to the NANPx6 peptide, the in vitro inhibition of sporozoite invasion activity, and kinetic parameters measured using intact mAbs or their Fab fragments. Buried surface area between mAb and its target epitope was also associated with in vivo protection. Association and disconnects between in vitro and in vivo readouts has important implications for the design and down-selection of the next generation of CSP based interventions.

Validation of a Novel Microscale Mold Patterning Protocol Based on Gelatin Methacrylate Photopolymerizable Hydrogels

2012 ◽  
Author(s):  
Paola Occhetta ◽  
Nasser Sadr ◽  
Francesco Piraino ◽  
Alberto Redaelli ◽  
Matteo Moretti ◽  
...  
Keyword(s):  
Uv Light ◽  
Three Dimensional ◽  
Light Irradiation ◽  
Uv Light Irradiation ◽  
Geometrical Features ◽  
3D Architecture ◽  
Pdms Replica ◽  
Size Scales

Native tissues are composed of functional three-dimensional (3D) units on the scale of 100–1000μm. The 3D architecture of these repeating units underlies the coordination of multicellular processes such as proliferation, differentiation, migration and apoptosis[1]. The requirement for 3D biomimetic matrices to mimic in vitro the ECM microarchitecture found in vivo becomes relevant in complex and vascularized tissue engineered models[2]. Among others, photopolymerizable hydrogels offer tunable geometrical features similar to the macromolecular-based components of soft ECM [3], can be crosslinked either in vivo or in vitro in the presence of a photoinitiator agent (PI) using visible or ultraviolet (UV) light irradiation, and have shown good compatibility with several protocols for cell embedding at different size-scales. In the present study, a new protocol to obtain cell-laden hydrogel micropatterns with highly controlled geometrical features is presented, based on the combination of polydimethylsiloxane (PDMS) replica molding and UV photopolimerization of methacrylate gelatin (GelMA).

scholarly journals cDNA display coupled with next-generation sequencing for rapid activity-based screening: Comprehensive analysis of transglutaminase substrate preference

2021 ◽  
Author(s):  
Jasmina Damnjanović ◽  
Nana Odake ◽  
Jicheng Fan ◽  
Beixi Jia ◽  
Takaaki Kojima ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Substrate Preference ◽  
Stable Complex ◽  
Next Generation ◽  
Library Size ◽  
Binary Model ◽  
Wide Range ◽  
Generation Sequencing ◽  
Selection Of

AbstractcDNA display is an in vitro display technology based on a covalent linkage between a protein and its corresponding mRNA/cDNA, where a stable complex is formed suitable for a wide range of selection conditions. A great advantage of cDNA display is the ability to handle enormous library size (1012) in a microtube scale, in a matter of days. To harness its benefits, we aimed at developing a platform which combines the advantages of cDNA display with high-throughput and accuracy of next-generation sequencing (NGS) for the selection of preferred substrate peptides of transglutaminase 2 (TG2), a protein cross-linking enzyme. After the optimization of the platform by the repeated screening of binary model libraries consisting of the substrate and non-substrate peptides at different ratios, screening and selection of combinatorial peptide library randomized at positions -1, +1, +2, and +3 from the glutamine residue was carried out. Enriched cDNA complexes were analyzed by NGS and bioinformatics, revealing the comprehensive amino acid preference of the TG2 at targeted positions of the peptide backbone. This is the first report on the cDNA display/NGS screening system to yield comprehensive data on TG substrate preference. Although some issues remain to be solved, this platform can be applied to the selection of other TGs and easily adjusted for the selection of other peptide substrates and even larger biomolecules.

Selection of DNA aptamer that specific binding human carcinoembryonic antigen in vitro

2007 ◽  
Vol 21 (5) ◽  
pp. 277-281 ◽  
Author(s):  
Lifeng Wang ◽  
Baorui Liu ◽  
Haitao Yin ◽  
Jia Wei ◽  
Xiaoping Qian ◽  
...  
Keyword(s):  
Carcinoembryonic Antigen ◽  
Specific Binding ◽  
Dna Aptamer ◽  
Selection Of

scholarly journals Phage Display Selection of an Anti-Idiotype-Antibody with Broad-Specificity to Deoxynivalenol Mycotoxins

Toxins ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 18
Author(s):  
Janne Leivo ◽  
Markus Vehniäinen ◽  
Urpo Lamminmäki
Keyword(s):  
In Vitro Selection ◽  
Specific Binding ◽  
Binding Properties ◽  
Phage Displays ◽  
Synthetic Antibody ◽  
Wide Range ◽  
Antibody Libraries ◽  
Rapid Generation ◽  
Selection Of

The use of synthetic antibody libraries and phage displays provides an efficient and robust method for the generation of antibodies against a wide range of targets with highly specific binding properties. As the in vitro selection conditions can be easily controlled, these methods enable the rapid generation of binders against difficult targets such as toxins and haptens. In this study, we used deoxynivalenol mycotoxin as a target to generate anti-idiotype-antibodies with unique binding properties from synthetic antibody libraries. The binding of the selected anti-idiotype antibodies can be efficiently inhibited with the addition of free isoforms of deoxynivalenol. The antibody was consecutively used to develop deoxynivalenol-specific ELISA and TRF-immunoassays, which can detect deoxynivalenol and two of the most common metabolic isoforms in the range of 78–115 ng/mL.

Inactivation of Protozoan Parasites in Food, Water, and Environmental Systems

2006 ◽  
Vol 69 (11) ◽  
pp. 2786-2808 ◽  
Author(s):  
MARILYN C. ERICKSON ◽  
YNES R. ORTEGA
Keyword(s):  
Uv Light ◽  
Synergistic Effects ◽  
Storage Conditions ◽  
Protozoan Parasites ◽  
Environmental Systems ◽  
Sequential Inactivation ◽  
Chemical Disinfectant ◽  
Physical Treatments ◽  
Selection Of

Protozoan parasites can survive under ambient and refrigerated storage conditions when associated with a range of substrates. Consequently, various treatments have been used to inactivate protozoan parasites (Giardia, Cryptosporidium, and Cyclospora) in food, water, and environmental systems. Physical treatments that affect survival or removal of protozoan parasites include freezing, heating, filtration, sedimentation, UV light, irradiation, high pressure, and ultrasound. Ozone is a more effective chemical disinfectant than chlorine or chlorine dioxide for inactivation of protozoan parasites in water systems. However, sequential inactivation treatments can optimize existing treatments through synergistic effects. Careful selection of methods to evaluate inactivation treatments is needed because many studies that have employed vital dye stains and in vitro excystation have produced underestimations of the effectiveness of these treatments.

scholarly journals A new strategy for the in vitro selection of stapled peptide inhibitors by mRNA display

2019 ◽  
Vol 55 (61) ◽  
pp. 8959-8962 ◽  
Author(s):  
Emil S. Iqbal ◽  
Stacie L. Richardson ◽  
Nicolas A. Abrigo ◽  
Kara K. Dods ◽  
H. Estheban Osorio Franco ◽  
...  
Keyword(s):  
In Vitro Selection ◽  
Peptide Inhibitors ◽  
Mrna Display ◽  
Stapled Peptide ◽  
New Strategy ◽  
The Creation ◽  
Selection Of

α-methyl Cys incorporation plus cyclization allows for the creation of α-helical libraries by mRNA display.

In vitro selection of peptide aptamers using a ribosome display for a conducting polymer

2014 ◽  
Vol 117 (4) ◽  
pp. 501-503 ◽  
Author(s):  
Zha Li ◽  
Takanori Uzawa ◽  
Haichao Zhao ◽  
Shyh-Chyang Luo ◽  
Hsiao-hua Yu ◽  
...  
Keyword(s):  
Conducting Polymer ◽  
In Vitro Selection ◽  
Ribosome Display ◽  
Peptide Aptamers ◽  
Selection Of

scholarly journals Methods to disinfect and decontaminate SARS-CoV-2: a systematic review of in vitro studies

2021 ◽  
Vol 8 ◽  
pp. 204993612199854
Author(s):  
Chun Shing Kwok ◽  
Mustafa Dashti ◽  
Jacopo Tafuro ◽  
Mojtaba Nasiri ◽  
Elena-Andra Muntean ◽  
...  
Keyword(s):  
Systematic Review ◽  
Povidone Iodine ◽  
Uv Light ◽  
Light Irradiation ◽  
Uv Light Irradiation ◽  
Iodine Complex ◽  
Chemical Agents ◽  
Hand Wash ◽  
Different Types

Background: Cleaning is a major control component for outbreaks of infection. However, for the SARS-CoV-2 pandemic, there is limited specific guidance regarding the proper disinfection methods that should be used. Methods: We conducted a systematic review of the literature on cleaning, disinfection or decontamination methods in the prevention of SARS-CoV-2. Results: A total of 27 studies were included, reporting a variety of methods with which the effectiveness of interventions were assessed. Virus was inoculated onto different types of material including masks, nasopharyngeal swabs, serum, laboratory plates and simulated saliva, tears or nasal fluid and then interventions were applied in an attempt to eliminate the virus including chemical, ultraviolet (UV) light irradiation, and heat and humidity. At body temperature (37°C) there is evidence that the virus will not be detectable after 2 days but this can be reduced to non-detection at 30 min at 56°C, 15 min at 65°C and 2 min at 98°C. Different experimental methods testing UV light have shown that it can inactivate the virus. Light of 254–365 nm has been used, including simulated sunlight. Many chemical agents including bleach, hand sanitiser, hand wash, soap, ethanol, isopropanol, guandinium thiocynate/t-octylphenoxypolyethoxyethanol, formaldehyde, povidone-iodine, 0.05% chlorhexidine, 0.1% benzalkonium chloride, acidic electrolysed water, Clyraguard copper iodine complex and hydrogen peroxide vapour have been shown to disinfect SARS-CoV-2. Conclusions: Heating, UV light irradiation and chemicals can be used to inactivate SARS-CoV-2 but there is insufficient evidence to support one measure over others in clinical practice.

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