Responsive self-assembly is a general process in biological systems and is highly desired in engineered systems. DNA nanostructures provide a versatile molecular platform for studying such responsive self-assembly. Various triggers have been explored for DNA nanostructures. However, each trigger requires a unique mechanism for its response. This situation brings a great challenge to engineer the responsiveness. Herein, we propose an aptamer-based, allosteric mechanism for responsive DNA self-assembly. The aptamer–ligand binding causes the DNA motif to change its conformation and thus influences the motif assembly. With a model of an ATP aptamer, we have demonstrated the responsive assembly. Such responsive behavior, we believe, will be an important element for molecular machines, bioimaging/biosensing, and drug delivery.

An approach is established for the specific immobilization of GPCRs from cell lysates that circumvents labor intensive purification procedures and minimize loss of activity.

No abstract

Protein immobilization is particularly significant in proteomics, interactomics, and in vitro drug screening. It is an essential primary step for numerous biological techniques that rely on immobilized proteins with controlled orientation, high conformational stability, and high activity (CHH). These have challenged the current immobilization strategy and demanded increasing efforts for an efficient method to meet the CHH immobilization in a single step. Herein, we proposed a covalent inhibitorbased, one-step method for G protein-coupled receptor (GPCR) immobilization inspired by the covalent reaction between an epidermal growth factor receptor (EGFR)-tag and its inhibitor ibrutinib. We immobilized endothelin receptor A (ETA) containing a fusion EGFR tag onto an ibrutinib-coated macroporous silica gel. The immobilized ETA proved to have demonstrable ligand-binding activity and specificity, thus resulting in a chromatographic technology allowing receptor−ligand interaction analysis and lead identification. Such immobilization method is attractable, owing to the properties of mild reacting conditions, fast rate, high yield, and good stability of the conjugated protein. It will be applicable to biochips, biosensors, and biocatalysts.

The past decade has witnessed the great promise of strategies for ligand discovery based on surface-immobilized GPCRs. We present here a method for preparation of immobilized GPCRs. Key features include covalent immobilization with high specificity and robust application in drugreceptor interaction analysis and ligand screening. In our example assay using beta 2 -adrenergic receptor (β 2 -AR), the human DNA repair protein O 6alkylguanine-DNA alkyltransferase (hAGT) fusion receptor expressed in Escherichia coli was directly captured onto polyethylene glycol polyacrylamide (PEGA) resin. We observed even distribution and physiological functions of β 2 -AR on the resin. The immobilized β 2 -AR as a stationary phase enabled us to rapidly determine the binding of four drugs to β 2 -AR. By coupling this assay to mass spectrometry, we screened rosmarinic acid as a bioactive compound targeting β 2 -AR in Fructus Perillae. We concluded that O 6 -benzylguanine derivative-functionalized supporter is promising for specific immobilization of hAGT-tagged proteins; immobilized receptor chromatography has great potential in screening receptor-binding leads from herbal plants or traditional medicine recipes.

Owing to the promising clinical efficacy and relatively simple composition, Shuang-Huang-Lian prescription is widely prescribed for the treatment of acute upper respiratory tract infection and acute bronchitis in practice. This necessitates the understanding of the bioactive compounds of the prescription and their binding mechanism to β -adrenoceptor, which mediates the aforementioned ailments. In this work, a column containing immobilized β -adrenoceptor was prepared using a diazonium salt reaction. The bioactive compound collected from the β -adrenoceptor column was identified as chlorogenic acid by using high-performance liquid chromatography coupled with ion trap mass spectrometry. Using an injection amount dependent method, chlorogenic acid proved the binding to β -adrenoceptor through two kinds of sites. The numbers of the sites were (1.42 ± 0.03) × 10 and (9.06 ± 0.49) × 10 M. The association constants were (2.72 ± 0.01) × 10 and (2.80 ± 0.01) × 10 M , respectively. Molecular docking analysis of the interaction between chlorogenic acid and β -adrenoceptor indicated that the binding mainly occurred on Ser , Ser , and Phe of β -adrenoceptor. These results paved the way to screen bioactive compounds of other traditional medicines by receptor chromatography.

Allosteric ligands are promising drugs owing to their remote regulations of the orthosteric ligand signaling pathway. There are few allosteric ligands due to the lack of handy and efficacious method for the screening. Herein, we developed an affinity chromatographic method for allosteric ligand screening by immobilizing purified beta2 adrenoceptor (β2-AR) onto macroporous silica gel by a two-point tethering method. The method relies on the occupation of the orthosteric site by an antagonist and the chelation of N-terminal His-tag of the receptor and Ni2+ coated on the gel. The immobilized β2-AR demonstrated the greatest allosteric responsive feature when Cmpd-15 (0.25 μM) was included in the mobile phase. Under the same conditions, the association constants of three agonists (salbutamol, terbutaline, and tulobuterol) reduced to 47%, 19%, and 27% compared with the data without the inclusion of Cmpd-15 in the mobile phase. APF was screened as a potential allosteric modulator of β2-AR by applying the immobilized receptor in a natural product-derived DNA-encoded chemical library (DEL). Relying on these results, we reasoned that the current method has potential in screening allosteric ligands of the receptor. We expect that it is applicable for the discovery of new allosteric binding sites of a target protein and screening allosteric modulators of the other receptors from complex samples.

A new mathematical model and frontal analysis were used to characterize the binding behavior of caffeic acid to human serum albumin (HSA) based on high-performance affinity chromatography. The experiments were carried out by injecting various mole amounts of the drug onto an immobilized HSA column. They indicated that caffeic acid has only one type of binding site to HSA on which the association constant was 2.75 × 10(4) /m. The number of the binding site involving the interaction between caffeic acid and HSA was 69 nm. The data obtained by the frontal analysis appeared to present the same results for both the association constant and the number of binding sites. This new model based on the relationship between the mole amounts of injection and capacity factors assists understanding of drug-protein interaction. The proposed model also has the advantages of ligand saving and rapid operation.