scholarly journals Evolved polymerases facilitate selection of fully 2′-OMe-modified aptamers

2017 ◽  
Vol 8 (12) ◽  
pp. 8179-8182 ◽  
Author(s):  
Zhixia Liu ◽  
Tingjian Chen ◽  
Floyd E. Romesberg
Keyword(s):  
Dna Polymerases ◽  
High Affinity ◽  
Selection Of

Evolved DNA polymerases are used in selections with fully 2′-OMe modified libraries to identify aptamers with high affinity for HNE.

Combination of ribosome and phage display for fast selection of high affinity VHH antibody fragments

2019 ◽  
pp. 27-33
Author(s):  
YuE Kravchenko ◽  
SV Ivanov ◽  
DS Kravchenko ◽  
EI Frolova ◽  
SP Chumakov
Keyword(s):  
Phage Display ◽  
Selection Procedure ◽  
Free System ◽  
Phage Displays ◽  
High Affinity ◽  
Binding Domains ◽  
A Cell ◽  
Vhh Antibodies ◽  
Efficiency Of Selection ◽  
Selection Of

Selection of antibodies using phage display involves the preliminary cloning of the repertoire of sequences encoding antigen-binding domains into phagemid, which is considered the bottleneck of the method, limiting the resulting diversity of libraries and leading to the loss of poorly represented variants before the start of the selection procedure. Selection in cell-free conditions using a ribosomal display is devoid from this drawback, however is highly sensitive to PCR artifacts and the RNase contamination. The aim of the study was to test the efficiency of a combination of both methods, including pre-selection in a cell-free system to enrich the source library, followed by cloning and final selection using phage display. This approach may eliminate the shortcomings of each method and increase the efficiency of selection. For selection, alpaca VHH antibody sequences suitable for building an immune library were used due to the lack of VL domains. Analysis of immune libraries from the genes of the VH3, VHH3 and VH4 families showed that the VHH antibodies share in the VH3 and VH4 gene groups is insignificant, and selection from the combined library is less effective than from the VHH3 family of sequences. We found that the combination of ribosomal and phage displays leads to a higher enrichment of high-affinity fragments and avoids the loss of the original diversity during cloning. The combined method allowed us to obtain a greater number of different high-affinity sequences, and all the tested VHH fragments were able to specifically recognize the target, including the total protein extracts of cell cultures.

Precise selection of aptamers targeting PD-L1 positive small extracellular vesicles on magnetic chips

2021 ◽  
Vol 57 (29) ◽  
pp. 3555-3558
Author(s):  
Hao-Yu Dong ◽  
Qi-Hui Xie ◽  
Dai-Wen Pang ◽  
Gang Chen ◽  
Zhi-Ling Zhang
Keyword(s):  
Extracellular Vesicles ◽  
High Affinity ◽  
Selection Of

High affinity aptamers that target small extracellular vesicles displaying PD-L1 in its natural conformation were successfully selected.

In Vitro Selection of Oligonucleotide Acid Aptamers against Pathogenic Vibrio Alginolyticus by SELEX

2010 ◽  
Vol 439-440 ◽  
pp. 1456-1462 ◽  
Author(s):  
Jiang Zheng ◽  
Yu Bao Li ◽  
Jia Xiang Li ◽  
Jun Wang ◽  
Yong Quan Su
Keyword(s):  
Secondary Structures ◽  
Magnetic Bead ◽  
Vibrio Alginolyticus ◽  
Pathogenic Microorganism ◽  
Asymmetric Pcr ◽  
High Affinity ◽  
Amplification Method ◽  
Pathogenic Vibrio ◽  
Selection Of

Detection of pathogenic microorganism is very necessary in the control of infectious disease prevailing in aquiculture animals. However, most of the present techniques can not meet the need of the quick field inspection. Systematic evolution of ligands by exponential enrichment (SELEX) is a new molecular recognition way for generating high affinity oligonucleotide acid aptamers, a new nucleotide acid material, which have been widely used in the detections of proteins, cells and so on. In the present paper, the technology was applied to select the high affinity aptamers against pathogenic microorganism Vibrio alginolyticus, which could be used for the rapid field detection of the microorganism. Based on the designment of the ssDNA library of 76 nucleotide acids with 35-base random region, the SELEX system for the selection of the high affinity aptamers against Vibrio alginolyticus was established. In the SELEX system, asymmetric PCR was proved to be a better amplification method for the ssDNA library than the reported affinity magnetic bead method, and the corresponding parameters of the asymmetric PCR were also studied and optimized. The affinity of the final ssDNA library increased by nearly 200% compared with the original library. Cloning and sequencing of the final ssDNA library showed that there were at least two kinds of ssDNAs with different length in the affinity ssDNA library: one was 76 bases, another was 149 bases. Simulation of the secondary structures showed that the secondary structures of the two fragments were different greatly, suggesting that the two fragments could bind to different sites of V. alginolyticus surface.

scholarly journals DNA Polymerase: Structural Homology, Conformational Dynamics, and the Effects of Carcinogenic DNA Adducts

2010 ◽  
Vol 2010 ◽  
pp. 1-15 ◽  
Author(s):  
Richard G. Federley ◽  
Louis J. Romano
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Dna Adducts ◽  
Structural Changes ◽  
Dna Polymerases ◽  
Conformational Dynamics ◽  
Three Dimensional ◽  
Structural Homology ◽  
Human Right ◽  
Selection Of

DNA replication is vital for an organism to proliferate and lying at the heart of this process is the enzyme DNA polymerase. Most DNA polymerases have a similar three dimensional fold, akin to a human right hand, despite differences in sequence homology. This structural homology would predict a relatively unvarying mechanism for DNA synthesis yet various polymerases exhibit markedly different properties on similar substrates, indicative of each type of polymerase being prescribed to a specific role in DNA replication. Several key conformational steps, discrete states, and structural moieties have been identified that contribute to the array of properties the polymerases exhibit. The ability of carcinogenic adducts to interfere with conformational processes by directly interacting with the protein explicates the mutagenic consequences these adducts impose. Recent studies have identified novel states that have been hypothesised to test the fit of the nascent base pair, and have also shown the enzyme to possess a lively quality by continually sampling various conformations. This review focuses on the homologous structural changes that take place in various DNA polymerases, both replicative and those involved in adduct bypass, the role these changes play in selection of a correct substrate, and how the presence of bulky carcinogenic adducts affects these changes.

Selection of high affinity RNA ligands to the bacteriophage R17 coat protein

1992 ◽  
Vol 228 (3) ◽  
pp. 862-869 ◽  
Author(s):  
Dan Schneider ◽  
Craig Tuerk ◽  
Larry Gold
Keyword(s):  
Coat Protein ◽  
High Affinity ◽  
Rna Ligands ◽  
Selection Of
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