Quantitative synthesis of protein–DNA conjugates with 1 : 1 stoichiometry

Yan, Xiaowen; Zhang, Hongquan; Wang, Zhixin; Peng, Hanyong; Tao, Jeffrey; Li, Xing‐Fang; Le, X. Chris

doi:10.1039/c8cc03268h

Cited by 22 publications

(22 citation statements)

References 37 publications

(40 reference statements)

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“…Another utility of SENDR was also demonstrated in the formation of DNA-protein conjugates, which are becoming increasingly valuable in the production of long-acting and/or targeted oligonucleotide drugs. [47][48][49][50] An oligonucleotide was ligated by SENDR (using Ψ-module 6) with an activated disulfide group resulting in 52 (Figure 6A). This construct could be used directly, without purification in a disulfide forming reaction with bovine serum albumin (BSA).…”

Section: Sendr: Dna-protein Conjugatesmentioning

confidence: 99%

Synthetic Elaboration of Native DNA by RASS (SENDR)

Flood¹,

Knouse²,

Vantourout³

et al. 2020

Preprint

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The controlled, site-specific ligation of molecules to native DNA remains an unanswered challenge. Herein, we report a simple solution to achieve this ligation through the tactical combination of two recently developed technologies: One for the manipulation of DNA in organic media, and another for the chemoselective labeling of alcohols. Reversible Adsorption of Solid Support (RASS) is employed to immobilize DNA and facilitate its transfer into dry acetonitrile. Subsequent ligation with P(V)-based Ψ reagents takes place in high yield with exquisite selectivity for the exposed 3’ or 5’ alcohols on DNA. This two-stage process, dubbed SENDR for Synthetic Elaboration of Native DNA by RASS, can be applied to a multitude of DNA conformations and sequences with a variety of functionalized Ψ reagents to generate useful constructs. Such entities can address numerous longstanding challenges, including the selective single coupling of DNA to proteins, ASOs, and functional small molecules, and also can allow the synthesis of doubly-labeled congeners for novel probe constructs including ones of potential interest to COVID-19 research. Finally, a prototype for the industrialization of SENDR in a kit format is presented.

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Section: Sendr: Dna-protein Conjugatesmentioning

confidence: 99%

Synthetic Elaboration of Native DNA by RASS (SENDR)

Flood¹,

Knouse²,

Vantourout³

et al. 2020

Preprint

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“…Affinity‐guided conjugation for creation of protein–DNA conjugates is known as DNA‐templated protein conjugation (DPTC) as illustrated in Figure . In this method, a guiding strand (blue) is modified with an affinity moiety and forms a complex with the protein.…”

Section: Label‐containing Moietymentioning

confidence: 99%

“…Generally, very few highly reactive electrophiles have been used for affinity‐guided conjugation. However, for DNA templated protein conjugation, it has been demonstrated that by lowering the concentrations of the reactants, even N ‐hydroxysuccinimide (NHS) esters can be utilized for affinity‐guided conjugation (Figure C) . The NHS esters have been employed with ligand‐, metal‐, and aptamer‐ guided approaches.…”

Section: Reactive Moietymentioning

confidence: 99%

“…However, for DNA templated protein conjugation, it has been demonstrated that by lowering the concentrations of the reactants, even N ‐hydroxysuccinimide (NHS) esters can be utilized for affinity‐guided conjugation (Figure C) . The NHS esters have been employed with ligand‐, metal‐, and aptamer‐ guided approaches. Furthermore, it was demonstrated that the NHS esters could be used for modification of proteins on the surface of live cells …”

Section: Reactive Moietymentioning

confidence: 99%

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Considerations on Probe Design for Affinity‐Guided Protein Conjugation

2019

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The plethora of methods developed for the creation of protein conjugates often differs significantly with regard to the heterogeneity of the resulting products, in the degree of genetic manipulation of the protein required, and in the technical skills required to perform the conjugation procedure. Affinity‐guided protein conjugation is a protein labeling methodology based on noncovalent binding interactions between a labeling probe and the protein of interest. These interactions increase the local concentration of a reactive group in the probe on the protein surface thus facilitating the conjugation in proximity of the complexation site. The ability to produce high‐quality conjugates from nongenetically modified proteins both in vitro, but also in cells, demonstrates the power of affinity‐guided protein conjugation. Here, we present the progress of affinity‐guided protein conjugation in relation to selective protein labeling in living systems and the formation of high‐quality protein conjugates. Furthermore, the probe design will be discussed in relation to the utility of the probe for labeling in vitro or in living systems.

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“…With regard to site-specific modifications, several methods are reported utilizing unnatural amino acids [ 6 ], intein [ 10 ], enzyme-catalyzed tags [ 11 ], self-labeling enzymes [ 12 ], or DNA-templated conjugation [ 13 , 14 ]. However, additional materials other than the DNA and protein are required to perform these conjugation methods.…”

Section: Introductionmentioning

confidence: 99%

Application of a Glucose Dehydrogenase-Fused with Zinc Finger Protein to Label DNA Aptamers for the Electrochemical Detection of VEGF

Lee

Tatsumi

Tsukakoshi

et al. 2020

Sensors

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Aptamer-based electrochemical sensors have gained attention in the context of developing a diagnostic biomarker detection method because of their rapid response, miniaturization ability, stability, and design flexibility. In such detection systems, enzymes are often used as labels to amplify the electrochemical signal. We have focused on glucose dehydrogenase (GDH) as a labeling enzyme for electrochemical detection owing to its high enzymatic activity, availability, and well-established electrochemical principle and platform. However, it is difficult and laborious to obtain one to one labeling of a GDH-aptamer complex with conventional chemical conjugation methods. In this study, we used GDH that was genetically fused to a DNA binding protein, i.e., zinc finger protein (ZF). Fused GDH can be attached to an aptamer spontaneously and site specifically in a buffer by exploiting the sequence-specific binding ability of ZF. Using such a fusion protein, we labeled a vascular endothelial growth factor (VEGF)-binding aptamer with GDH and detected the target electrochemically. As a result, upon the addition of glucose, the GDH labeled on the aptamer generated an amperometric signal, and the current response increased dependent on the VEGF concentration. Eventually, the developed electrochemical sensor proved to detect VEGF levels as low as 105 pM, thereby successfully demonstrating the concept of using ZF-fused GDH to enzymatically label aptamers.

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Quantitative synthesis of protein–DNA conjugates with 1 : 1 stoichiometry

Cited by 22 publications

References 37 publications

Synthetic Elaboration of Native DNA by RASS (SENDR)

Synthetic Elaboration of Native DNA by RASS (SENDR)

Considerations on Probe Design for Affinity‐Guided Protein Conjugation

Application of a Glucose Dehydrogenase-Fused with Zinc Finger Protein to Label DNA Aptamers for the Electrochemical Detection of VEGF

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