scholarly journals RE-SELEX: Restriction Enzyme-Based Evolution of Structure-Switching Aptamer Biosensors

2021 ◽  
Author(s):  
Aimee A. Sanford ◽  
Alexandra E. Rangel ◽  
Trevor A. Feagin ◽  
Robert G. Lowery ◽  
Hector Argueta-Gonzalez ◽  
...  
Keyword(s):  
Conformational Change ◽  
Small Molecule ◽  
High Selectivity ◽  
Restriction Enzymes ◽  
Selection Methods ◽  
Recognition Elements ◽  
Evolution Of Structure ◽  
Significant Conformational Change ◽  
Structure Switching ◽  
General Method

<p><b>ABSTRACT </b></p> <p>Aptamers are widely employed as recognition elements in small molecule biosensors due to their ability to recognize small molecule targets with high affinity and selectivity. Structure-switching aptamers are particularly promising for biosensing applications because target-induced conformational change can be directly linked to an output. However, traditional evolution methods do not select for the significant conformational change needed to create structure-switching biosensors. Modified selection methods have been described to select for structure-switching architectures, but these remain limited by the need for immobilization. Herein we describe the first homogenous, structure-switching aptamer selection that directly reports on biosensor capacity for the target. We exploit the activity of restriction enzymes to isolate aptamer candidates that undergo target-induced displacement of a short complementary strand. As an initial demonstration of the utility of this approach, we performed selection against kanamycin A. Four enriched candidate sequences were successfully characterized as structure-switching biosensors for detection of kanamycin A. Optimization of biosensor conditions afforded facile detection of kanamycin A (90 µM – 10 mM) with high selectivity over three other aminoglycosides. This research demonstrates a general method to directly select for structure-switching biosensors and can be applied to a broad range of small molecule targets.</p>

scholarly journals RE-SELEX: Restriction Enzyme-Based Evolution of Structure-Switching Aptamer Biosensors

2021 ◽  
Author(s):  
Aimee A. Sanford ◽  
Alexandra E. Rangel ◽  
Trevor A. Feagin ◽  
Robert G. Lowery ◽  
Hector Argueta-Gonzalez ◽  
...  
Keyword(s):  
Conformational Change ◽  
Small Molecule ◽  
High Selectivity ◽  
Restriction Enzymes ◽  
Selection Methods ◽  
Recognition Elements ◽  
Evolution Of Structure ◽  
Significant Conformational Change ◽  
Structure Switching ◽  
General Method

<p><b>ABSTRACT </b></p> <p>Aptamers are widely employed as recognition elements in small molecule biosensors due to their ability to recognize small molecule targets with high affinity and selectivity. Structure-switching aptamers are particularly promising for biosensing applications because target-induced conformational change can be directly linked to an output. However, traditional evolution methods do not select for the significant conformational change needed to create structure-switching biosensors. Modified selection methods have been described to select for structure-switching architectures, but these remain limited by the need for immobilization. Herein we describe the first homogenous, structure-switching aptamer selection that directly reports on biosensor capacity for the target. We exploit the activity of restriction enzymes to isolate aptamer candidates that undergo target-induced displacement of a short complementary strand. As an initial demonstration of the utility of this approach, we performed selection against kanamycin A. Four enriched candidate sequences were successfully characterized as structure-switching biosensors for detection of kanamycin A. Optimization of biosensor conditions afforded facile detection of kanamycin A (90 µM – 10 mM) with high selectivity over three other aminoglycosides. This research demonstrates a general method to directly select for structure-switching biosensors and can be applied to a broad range of small molecule targets.</p>

scholarly journals RE-SELEX: Restriction Enzyme-Based Evolution of Structure-Switching Aptamer Biosensors

2021 ◽  
Author(s):  
Aimee A. Sanford ◽  
Alexandra E. Rangel ◽  
Trevor A. Feagin ◽  
Robert G. Lowery ◽  
Hector Argueta-Gonzalez ◽  
...  
Keyword(s):  
Conformational Change ◽  
Small Molecule ◽  
High Selectivity ◽  
Restriction Enzymes ◽  
Selection Methods ◽  
Recognition Elements ◽  
Evolution Of Structure ◽  
Significant Conformational Change ◽  
Structure Switching ◽  
General Method

<p><b>ABSTRACT </b></p> <p>Aptamers are widely employed as recognition elements in small molecule biosensors due to their ability to recognize small molecule targets with high affinity and selectivity. Structure-switching aptamers are particularly promising for biosensing applications because target-induced conformational change can be directly linked to an output. However, traditional evolution methods do not select for the significant conformational change needed to create structure-switching biosensors. Modified selection methods have been described to select for structure-switching architectures, but these remain limited by the need for immobilization. Herein we describe the first homogenous, structure-switching aptamer selection that directly reports on biosensor capacity for the target. We exploit the activity of restriction enzymes to isolate aptamer candidates that undergo target-induced displacement of a short complementary strand. As an initial demonstration of the utility of this approach, we performed selection against kanamycin A. Four enriched candidate sequences were successfully characterized as structure-switching biosensors for detection of kanamycin A. Optimization of biosensor conditions afforded facile detection of kanamycin A (90 µM – 10 mM) with high selectivity over three other aminoglycosides. This research demonstrates a general method to directly select for structure-switching biosensors and can be applied to a broad range of small molecule targets.</p>

scholarly journals RE-SELEX: Restriction Enzyme-Based Evolution of Structure-Switching Aptamer Biosensors 

2021 ◽  
Author(s):  
Aimee Alice Sanford ◽  
Alexandra E Rangel ◽  
Trevor A Feagin ◽  
Robert G Lowery ◽  
Hector S Argueta-Gonzalez ◽  
...  
Keyword(s):  
Small Molecule ◽  
Restriction Enzyme ◽  
High Affinity ◽  
Recognition Elements ◽  
Evolution Of Structure ◽  
Structure Switching

Aptamers are widely employed as recognition elements in small molecule biosensors due to their ability to recognize small molecule targets with high affinity and selectivity. Structure-switching aptamers are particularly promising...

A general method of isolating high molecular weight DNA from methanogenic archaea (archaebacteria)

1992 ◽  
Vol 38 (1) ◽  
pp. 65-68 ◽  
Author(s):  
Ken F. Jarrell ◽  
David Faguy ◽  
Anne M. Hebert ◽  
Martin L. Kalmokoff
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Thermal Denaturation ◽  
Dna Isolation ◽  
Methanogenic Archaea ◽  
Restriction Enzymes ◽  
Mole Percent ◽  
High Molecular Weight Dna ◽  
Isolation Methods ◽  
General Method

High molecular weight DNA was readily isolated from all methanogens treated, as well as from thermophilic anaerobic eubacteria, by grinding cells frozen in liquid N2, prior to lysis with SDS. DNA can subsequently be purified by the usual phenol–chloroform extractions. The procedure yields DNA readily cut by restriction enzymes and suitable for oligonucleotide probing, as well as for mole percent G + C content determination by thermal denaturation. The method routinely yields DNA of high molecular weight and is an improvement over DNA isolation methods for many methanogens, which often involve an initial breakage of the cells in a French pressure cell. Key words: methanogens, archaebacteria, archaea, DNA isolation.

scholarly journals Introducing structure-switching functionality into small-molecule-binding aptamers via nuclease-directed truncation

2018 ◽  
Vol 46 (13) ◽  
pp. e81-e81 ◽  
Author(s):  
Zongwen Wang ◽  
Haixiang Yu ◽  
Juan Canoura ◽  
Yingzhu Liu ◽  
Obtin Alkhamis ◽  
...  
Keyword(s):  
Small Molecule ◽  
Structure Switching

Metal–organic frameworks constructed from tib and carboxylate acid ligands: selective sensing of nitro explosives and magnetic properties

2017 ◽  
Vol 46 (23) ◽  
pp. 7567-7576 ◽  
Author(s):  
Liu Yang ◽  
Li Cao ◽  
Xiao Li ◽  
Chao Qin ◽  
Liang Zhao ◽  
...  
Keyword(s):  
Magnetic Properties ◽  
Small Molecule ◽  
High Selectivity ◽  
Metal Organic Frameworks ◽  
Fluorescent Sensors ◽  
Nitro Explosives ◽  
Metal Organic

Four novel metal–organic frameworks have been synthesized under certain conditions. 1 and 2 can be used as fluorescent sensors for small-molecule sensing with high selectivity. Meanwhile, 3 and 4 are both antiferromagnetic substances.

Discovery of Selective Small Molecule Pan-Pim Kinase Inhibitors with Potent Oral Efficacy in Murine Xenograft Models.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1726-1726
Author(s):  
Sean O'Brien ◽  
Mustapha Haddach ◽  
Cosmin Borsan ◽  
Jerome Michaux ◽  
Pauline Kerdoncuff ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Selectivity ◽  
Conflicts Of Interest ◽  
B Cell Lymphoma ◽  
Myelogenous Leukemia ◽  
Human Prostate ◽  
Prostate Tumors ◽  
Xenograft Models ◽  
Threonine Kinases

Abstract Abstract 1726 Poster Board I-752 The PIM family of serine/threonine kinases are pro-proliferative kinases activated by multiple cytokines and growth factor signaling. The Pim kinases are unusual in that they are regulated primarily by transcription and not by membrane recruitment or phosphorylation like other serine/threonine kinases. Activated cytokine receptors recruit JAKs to induce STAT-dependent transcription of the Pim genes. They are proto-oncogenes and have been implicated in the process of lymphomagenesis and malignant transformation. Pim overexpression has been reported in diffuse B cell lymphoma, chronic lymphocytic leukemia, FLT3-mediated acute myelogenous leukemia and prostate cancer. Pim-2 is over expressed in leukemias and lymphomas, whereas Pim-3 overexpression has been observed in melanoma, pancreatic and gastric tumors. The recent reports of elevated levels of Pim-1 expression in human prostate tumor biopsies implicate the Pim family of protein kinases in the progression of human prostate tumors. Further, in transgenic animal models, Pim-1 expression has been shown to be elevated in prostate tumors that are caused by overexpression of the c-myc oncogene. Recent evidence reveals the overlapping and compensatory nature of Pim-1 and Pim-2 phosphorylation and highlights the importance of inhibiting all isoforms. The emerging role of the PIM kinase family in hematological malignancies and solid tumors and the druggable nature of their ATP binding pocket make them attractive targets for anticancer drug development Utilizing a highly distinct molecular scaffold, CX-6258 was developed as a selective and potent small molecule pan-PIM kinase inhibitor. CX-6258 inhibits Pims 1, 2 and 3 with IC50 values in the low nanomolar range and high selectivity as evidenced in a screening panel of over 100 kinases. CX-6258 demonstrates potent in vitro antiproliferative activity, particularly in leukemia derived cell lines expressing the FLT3-ITD. Moreover, CX-6258 inhibits the phosphorylation of BAD and 4EBP1, known substrates for PIMs 1, 2 and 3. When delivered orally, this pan-Pim inhibitor is well tolerated and demonstrates potent antitumor activity in murine xenograft models of PIM driven cancer. Using CX-6258 as our “path finder” molecule, we have created four additional unique chemical scaffolds as pan-Pim inhibitors, and certain of these scaffolds can inhibit Pims 1, 2 and 3 in the picomolar range while exhibiting no inhibitory activity of the Flt3 protein kinase. The in vivo and in vitro profiles of these chemically diverse series are indicative of an effective and potent anti-cancer mechanism mediated through the selective inhibition of PIM kinase activity. Together, these findings exemplify that we have created multiple proprietary chemical series of pan-Pim inhibitors exhibiting picomolar potency and high selectivity. Disclosures No relevant conflicts of interest to declare.

The Discovery of Direct Noncovalent Bonded Interaction between Keap1 and Diethyl Maleate through Molecular Dynamics Simulations

2012 ◽  
Vol 192 ◽  
pp. 260-265 ◽  
Author(s):  
Xiu Li Lu ◽  
Shu Chao Chen ◽  
Yong Zhang ◽  
Li Zhang ◽  
Hong Sheng Liu ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Conformational Change ◽  
Md Simulations ◽  
Normal Function ◽  
Control Group ◽  
Diethyl Maleate ◽  
Btb Domain ◽  
Short Period ◽  
Significant Conformational Change ◽  
Dynamics Simulations

Keap1 negatively regulates the function of Nrf2 that is a major activator of genes encoding phase 2 detoxifying enzymes via sequestering cytoplasmic Nrf2 and subsequent degradation through the proteasome system. Reactive cysteine residues of Keap1 could be modified by Michael reaction acceptor molecules. Previous studies have shown that adduction at Cys151 by diethyl maleate (DEM) can give rise to a significant conformational change in Keap1 that leads to the dissociation of Keap1 from CUL3, hence inhibits Nrf2 ubiquitylation. The BTB domain of Keap1 plays a crucial role in both forming self-dimerization and binding to CUL3. In order to better understanding the molecular mechanism how DEM interact with amino acid residues around Cys151, we performed two molecular dynamics (MD) simulations including Keap1-DEM complex and Keap1 alone (control group). Interestingly, we found that after a short period of lingering around Cys151, DEM ultimately stabilized in a gap between two specific helixes away from the cavity around Cys151 and induced a concomitant significant conformational change of BTB domain of Keap1. Similar phenomenon, however, was not observed in the control group. These results suggested that DEM could impair the normal function of Keap1 by inducing the conformational change of BTB domain via direct noncovalent bonded interaction. Our research provides a new insight into another way of interaction between Keap1 and DEM in spite of their known Michael addition reaction, by which novel phase2 enzyme inducer drugs with higher specificity could be discovered in the future

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