scholarly journals Aptamers Against the β-Conglutin Allergen: Insights into the Behavior of the Shortest Multimeric(Intra)Molecular DNA G-Quadruplex

2021 ◽  
Vol 22 (3) ◽  
pp. 1150
Author(s):  
Ciara K. O’ Sullivan ◽  
Teresa Mairal ◽  
Miriam Jauset-Rubio ◽  
Marketa Svobodova ◽  
Vasso Skouridou ◽  
...  
Keyword(s):  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Time Of Flight ◽  
Higher Order ◽  
Binding Ability ◽  
Ionization Time ◽  
Flight Mass Spectrometry ◽  
G Quadruplex ◽  
Derivatives Of ◽  
Order Structures

In previous work, a 93-mer aptamer was selected against the anaphylactic allergen, β-conglutin and truncated to an 11-mer, improving the affinity by two orders of magnitude, whilst maintaining the specificity. This 11-mer was observed to fold in a G-quadruplex, and preliminary results indicated the existence of a combination of monomeric and higher-order structures. Building on this previous work, in the current study, we aimed to elucidate a deeper understanding of the structural forms of this 11-mer and the effect of the structure on its binding ability. A battery of techniques including polyacrylamide gel electrophoresis, high-performance liquid chromatography in combination with electrospray ionization time-of-flight mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight, thermal binding analysis, circular dichroism and nuclear magnetic resonance were used to probe the structure of both the 11-mer and the 11-mer flanked with TT- at either the 5′ or 3′ end or at both ends. The TT-tail at the 5′ end hinders stacking effects and effectively enforces the 11-mer to maintain a monomeric form. The 11-mer and the TT- derivatives of the 11-mer were also evaluated for their ability to bind its cognate target using microscale thermophoresis and surface plasmon resonance, and biolayer interferometry confirmed the nanomolar affinity of the 11-mer. All the techniques utilized confirmed that the 11-mer was found to exist in a combination of monomeric and higher-order structures, and that independent of the structural form present, nanomolar affinity was observed.

scholarly journals Comparative Proteomics Analysis of Phosphine-Resistant and Phosphine-Susceptible Sitophilus oryzae (Coleoptera: Curculionidae)

2021 ◽  
Vol 11 (9) ◽  
pp. 4163
Author(s):  
Hyun-Na Koo ◽  
Seung Ju Seok ◽  
Hyun Kyung Kim ◽  
Gil-Hah Kim ◽  
Jeong Oh Yang
Keyword(s):  
Molecular Mechanisms ◽  
Citrate Synthase ◽  
Expression Profiles ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sitophilus Oryzae ◽  
Time Of Flight ◽  
Ionization Time ◽  
Flight Mass Spectrometry ◽  
Phosphine Resistance ◽  
Protein Expression Profiles

A proteomic method combining two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF) was used to compare the protein expression profiles of phosphine-resistant (PH3-R) and -susceptible (PH3-S) strains of Sitophilus oryzae. Thirty-nine differentially expressed protein spots were identified between the PH3-R and PH3-S strains; 20 protein spots were upregulated, and 19 protein spots were downregulated in the PH3-R strain compared with their expression in the PH3-S strain. In particular, cytochrome oxidase subunit I showed 15-fold higher expression in the PH3-R strain than in the PH3-S strain. Additionally, citrate synthase 2, delta-1-pyrolline-5-carboxylate dehydrogenase, and triose-phosphate isomerase were highly expressed in the PH3-R strain. In summary, our study has improved understanding of the molecular mechanisms of phosphine resistance in the rice weevil.

scholarly journals Proteins Associated with Culex nigripalpus Nucleopolyhedrovirus Occluded Virions

2007 ◽  
Vol 81 (9) ◽  
pp. 4585-4590 ◽  
Author(s):  
Omaththage Perera ◽  
Terry B. Green ◽  
Stanley M. Stevens ◽  
Susan White ◽  
James J. Becnel
Keyword(s):  
Mass Spectrometry ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Time Of Flight ◽  
Open Reading Frames ◽  
Structural Proteins ◽  
Ionization Time ◽  
Protein Databases ◽  
Flight Mass Spectrometry ◽  
Culex Nigripalpus

ABSTRACT Occlusion-derived virions (ODVs) of the nucleopolyhedrovirus of Culex nigripalpus (CuniNPV) were purified by Ludox density gradient ultracentrifugation, and the proteins were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were identified by using Edman sequencing, matrix-assisted laser desorption ionization-time of flight mass spectrometry, nanoelectrospray quadrupole time-of-flight mass spectrometry, or a combination of these methods. Half of the 44 polypeptide sequences identified in this analysis were unique open reading frames (ORFs) encoded by the CuniNPV genome and did not show similarity to any other sequences present in protein databases. Of the 22 polypeptides that showed similarities to other baculovirus-encoded proteins, only 17 sequences have previously been identified as structural proteins. The newly identified CuniNPV structural proteins cun058, cun059, cun087, cun106, and cun109 are homologues of Autographa californica nucleopolyhedrovirus (AcMNPV) ORFs 68, 62, 98, 81, and 2, respectively. The products of four genes, namely, lef-1 (cun045), alkaline exonuclease (cun054), helicase (cun089), and DNA polymerase (cun091), were not detected in the CuniNPV ODV preparations. These four genes are conserved among all annotated baculovirus genomes, and their homologues have been detected in the ODV of AcMNPV.

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