Unraveling Determinants of Affinity Enhancement in Dimeric Aptamers for a Dimeric Protein

Manochehry, Sepehr; McConnell, Erin M.; Li, Yingfu

doi:10.1038/s41598-019-54005-4

Cited by 27 publications

(30 citation statements)

References 74 publications

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“…VEGF-binding properties of covalent V7t1 dimers were studied by EMSA experiments under non-denaturing conditions, which is a sensitive and widely used method to detect protein-nucleic acid interactions [61]. In fact, the electrophoretic mobility of an oligonucleotide is typically high when free in solution, and significantly retarded when complexed with its target protein [17,20,61]. In parallel experiments, both N.A.…”

Section: Binding Experiments With Vegf 165 Proteinmentioning

confidence: 99%

Tuning the Polymorphism of the Anti-VEGF G-rich V7t1 Aptamer by Covalent Dimeric Constructs

Riccardi

Musumeci

Platella

et al. 2020

IJMS

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In the optimization process of nucleic acid aptamers, increased affinity and/or activity are generally searched by exploring structural analogues of the lead compound. In many cases, promising results have been obtained by dimerization of the starting aptamer. Here we studied a focused set of covalent dimers of the G-quadruplex (G4) forming anti-Vascular Endothelial Growth Factor (VEGF) V7t1 aptamer with the aim of identifying derivatives with improved properties. In the design of these covalent dimers, connecting linkers of different chemical nature, maintaining the same polarity along the strand or inverting it, have been introduced. These dimeric aptamers have been investigated using several biophysical techniques to disclose the conformational behavior, molecularity and thermal stability of the structures formed in different buffers. This in-depth biophysical characterization revealed the formation of stable G4 structures, however in some cases accompanied by alternative tridimensional arrangements. When tested for their VEGF165 binding and antiproliferative activity in comparison with V7t1, these covalent dimers showed slightly lower binding ability to the target protein but similar if not slightly higher antiproliferative activity on human breast adenocarcinoma MCF-7 cells. These results provide useful information for the design of improved dimeric aptamers based on further optimization of the linker joining the two consecutive V7t1 sequences.

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Section: Binding Experiments With Vegf 165 Proteinmentioning

confidence: 99%

Tuning the Polymorphism of the Anti-VEGF G-rich V7t1 Aptamer by Covalent Dimeric Constructs

Riccardi

Musumeci

Platella

et al. 2020

IJMS

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“…extremities of this aptamer can play a key role in its optimal structuring for protein recognition, finally increasing its VEGF 165 -binding affinity. 189 Successively 190 More recently, the same research group performed a new in vitro selection of VEGF 165 -binding DNA aptamers by using a multistream selection strategy in which the concentration of the target protein was varied. After 11 rounds of selection by next-generation sequencing for every round in each stream and sequence analysis, they found some DNA aptamers already known, but also novel sequences.…”

Section: Rnv66mentioning

confidence: 99%

“…Among the analysed covalent dimers, the oligonucleotide with the highest affinity for VEGF 165 proved to be bisV7t1TEG2D, in which the inversion of polarity probably promotes a different three-dimensional arrangement of the G4 structures composing the aptamer, which is preferred by the protein. 220 In addition, Manochehry et al 190 designed a series of dimeric aptamers based on the covalent connection of two heterodimeric or homodimeric DNA sequences using thymidine-based linkers of different length. All the obtained dimers were studied in their affinity for VEGF 165 by EMSA assays.…”

Section: Dimeric and Multimeric Anti-vegf Dna-based Aptamersmentioning

confidence: 99%

“…While no affinity improvementcompared to the parent monomers-was observed with the heterodimeric aptamers, the designed homodimers showed significant affinity enhancement. 190 In particular, within this series, the best candidates in terms of binding 14 nM for the homodimer vs. 40 nM for the monomer; Table 5), as determined by EMSA analysis. The second valuable construct was the homodimer based on two +5′G + 3′C sequences, covalently connected through 20 thymidine residues, showing K d value around 6 nM (Table 5), slightly improved compared to the monomer analysed under the same conditions (K d = 10 nM).…”

Section: Dimeric and Multimeric Anti-vegf Dna-based Aptamersmentioning

confidence: 99%

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Anti‐VEGF DNA‐based aptamers in cancer therapeutics and diagnostics

Riccardi

Napolitano

Platella

et al. 2020

Medicinal Research Reviews

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The vascular endothelial growth factor (VEGF) family and its receptors play fundamental roles not only in physiological but also in pathological angiogenesis, characteristic of cancer progression. Aiming at finding putative treatments for several malignancies, various small molecules, antibodies, or protein‐based drugs have been evaluated in vitro and in vivo as VEGF inhibitors, providing efficient agents approved for clinical use. Due to the high clinical importance of VEGF, also a great number of anti‐VEGF nucleic acid‐based aptamers—that is, oligonucleotides able to bind with high affinity and specificity a selected biological target—have been developed as promising agents in anticancer strategies. Notable research efforts have been made in optimization processes of the identified aptamers, searching for increased target affinity and/or bioactivity by exploring structural analogues of the lead compounds. This review is focused on recent studies devoted to the development of DNA‐based aptamers designed to target VEGF. Their therapeutic potential as well as their significance in the construction of highly selective biosensors is here discussed.

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“…And the great dissociation constant (Kd) values of the VEGF 165 and CEA aptamers were indeed already demonstrated, with a Kd value of 37.9 nM for VEGF 165 and 5 nM for CEA. 39 , 43 , 44 At the same time, on the basis of the aptamers reported in the previous literatures, we added 10 T-base linkers at the 5ʹ, which can effectively reduce steric hindrance and facilitate the binding of aptamers to the target.…”

Section: Introductionmentioning

confidence: 99%

<p>Simultaneous Detection of VEGF and CEA by Time-Resolved Chemiluminescence Enzyme-Linked Aptamer Assay</p>

Mu¹,

Dong²,

Wang³

et al. 2020

IJN

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Background As two important tumor markers, vascular endothelial growth factor (VEGF) and carcinoembryonic antigen (CEA) have a great value for clinical application in the early diagnosis of cancer. Due to the complex composition of biological samples, the results from combined detection of CEA and VEGF are often taken as a comprehensive indicator in order to make an accurate judgment on a disease. However, most of the current methods can only be used to detect the content of one biomarker. Therefore, it is necessary to explore a simple, rapid, low-cost, and highly sensitive method for the simultaneous detection of CEA and VEGF. Methods Based on specific aptamers and magnetic separation, a time-resolved chemiluminescence enzyme-linked aptamer assay was developed for the simultaneous detections of CEA and VEGF in serum samples. Results Under the optimal conditions, the linear range of the calibration curve for VEGF was from 0.5 to 80 ng mL −1 , and the limit of detection was 0.1 ng mL −1 . The linear range of the calibration curve for CEA was 0.5 to 160 ng mL −1 , and the limit of detection was 0.1 ng mL −1 . The established method was applied to detect VEGF and CEA in serum samples. The results were consistent with those of commercial kits. Conclusion The method has high sensitivity and can quickly obtain accurate results, which could greatly improve the measurement efficiency, reduce the cost, and also reduce the volume of sample consumed. It can be seen that the method established in this study has important application value and broad application prospect in clinical diagnosis.

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Unraveling Determinants of Affinity Enhancement in Dimeric Aptamers for a Dimeric Protein

Cited by 27 publications

References 74 publications

Tuning the Polymorphism of the Anti-VEGF G-rich V7t1 Aptamer by Covalent Dimeric Constructs

Tuning the Polymorphism of the Anti-VEGF G-rich V7t1 Aptamer by Covalent Dimeric Constructs

Anti‐VEGF DNA‐based aptamers in cancer therapeutics and diagnostics

<p>Simultaneous Detection of VEGF and CEA by Time-Resolved Chemiluminescence Enzyme-Linked Aptamer Assay</p>

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