Energy Landscape of Aptamer/Protein Complexes Studied by Single‐Molecule Force Spectroscopy

Aptamers are single‐stranded nucleic acid molecules selected in vitro to bind to a variety of target molecules. Aptamers bound to proteins are emerging as a new class of molecules that rival commonly used antibodies in both therapeutic and diagnostic applications. With the increasing application of aptamers as molecular probes for protein recognition, it is important to understand the molecular mechanism of aptamer–protein interaction. Recently, we developed a method of using atomic force microscopy (AFM) to study the single‐molecule rupture force of aptamer/protein complexes. In this work, we investigate further the unbinding dynamics of aptamer/protein complexes and their dissociation‐energy landscape by AFM. The dependence of single‐molecule force on the AFM loading rate was plotted for three aptamer/protein complexes and their dissociation rate constants, and other parameters characterizing their dissociation pathways were obtained. Furthermore, the single‐molecule force spectra of three aptamer/protein complexes were compared to those of the corresponding antibody/protein complexes in the same loading‐rate range. The results revealed two activation barriers and one intermediate state in the unbinding process of aptamer/protein complexes, which is different from the energy landscape of antibody/protein complexes. The results provide new information for the study of aptamer–protein interaction at the molecular level.     

The Construction of DNA Logic Gates Restricted to Certain Live Cells Based on the Structure Programmability and Aptamer-Cell Affinity of G-Quadruplexes

DNA computation is considered a fascinating alternative to silicon-based computers; it has evoked substantial attention and made rapid advances. Besides realizing versatile functions, implementing spatiotemporal control of logic operations, especially at the cellular level, is also of great significance to the development of DNA computation. However, developing simple and efficient methods to restrict DNA logic gates performing in live cells is still a challenge. In this work, a series of DNA logic gates was designed by taking full advantage of the diversity and programmability of the G-quadruplex (G4) structure. More importantly, by further using the high affinity and specific endocytosis of cells to aptamer G4, an INHIBIT logic gate has been realized whose operational site is precisely restricted to specific live cells. The design strategy might have great potential in the field of molecular computation and smart bio-applications.     

Protein-Mediated Suppression of Rolling Circle Amplification for Biosensing with an Aptamer-Containing DNA Primer

We report a method to detect proteins via suppression of rolling circle amplification (RCA) by using an appropriate aptamer as the linear primer (denoted as an aptaprimer) to initiate RCA. In the absence of a protein target, the aptaprimer is free to initiate RCA, which can produce long DNA products that are detected via binding of a fluorescent intercalating dye. Introduction of a target causes the primer region within the aptamer to become unavailable for binding to the circular template, inhibiting RCA. Using SYBR Gold or QuantiFluor dyes as fluorescent probes to bind to the RCA reaction product, it is possible to produce a generic protein-modulated RCA assay system that does not require fluorophore- or biotin-modified DNA species, substantially reducing complexity and cost of reagents. Based on this modulation of RCA, we demonstrate the ability to produce both solution and paper-based assays for rapid and quantitative detection of proteins including platelet derived growth factor and thrombin.     

Graphene Oxide Protected Nucleic Acid Probes for Bioanalysis and Biomedicine

Recently, the binding ability of DNA on GO and resulting nuclease resistance have attracted increasing attention, leading to new applications both in vivo and in vitro. In vivo, nucleic acids absorbed on GO can be effectively protected from enzymatic degradation and biological interference in complicated samples, making it useful for targeted delivery, gene regulation, intracellular detection and imaging with high uptake efficiencies, high intracellular stability, and very low toxicity. In vitro, the adsorption of ssDNA on GO surface and desorption of dsDNA or well‐folded ssDNA from GO surface result in the protection and deprotection of DNA from nucleic digestion, respectively, which has led to target‐triggered cyclic enzymatic amplification methods (CEAM) for amplified detection of analytes with sensitivity 2–3 orders of magnitude higher than that of 1:1 binding strategies. This Concept article explores some of the latest developments in this field.     

Binding Characteristics Study of DNA based Aptamers for E. coli O157:H7

E. coli O157:H7 is a pathogenic bacterium producing verotoxins that could lead to serious complications such as hemolytic uremia syndrome. Fast detection of such pathogens is important. For rapid detection, aptamers are quickly gaining traction as alternative biorecognition molecules besides conventional antibodies. Several DNA aptamers have been selected for E. coli O157:H7. Nonetheless, there has not been a comparative study of the binding characteristics of these aptamers. In this work, we present a comprehensive analysis of binding characteristics including binding affinity (K_d) and binding capacity (B_max) of DNA-based aptamers for E. coli O157:H7 using qPCR. Our results show that aptamer E18R has the highest binding capacity to E. coli 157:H7 and the highest specificity over non-pathogenic E. coli strains K12 and DH5α. Our study also finds that the common biotin-tag modification at 5′ end typically changes the binding capacity significantly. For most of the selected aptamers, the binding capacity after a biotin-tag modification decreases. There exists a discrepancy in the binding capability between the selected aptamer and the aptamer used for detection. Our study also shows that a lower concentration of Mg²⁺ ions in the binding buffer leads to a decrease in the binding capacity of E17F and E18R, while it does not affect the binding capacity of S1 and EcoR1.     

DNA Nanoflowers for Multiplexed Cellular Imaging and Traceable Targeted Drug Delivery

We present a facile approach to make aptamer‐conjugated FRET (fluorescent resonance energy transfer) nanoflowers (NFs) through rolling circle replication for multiplexed cellular imaging and traceable targeted drug delivery. The NFs can exhibit multi‐fluorescence emissions by a single‐wavelength excitation as a result of the DNA matrix covalently incorporated with three dye molecules able to perform FRET. Compared with the conventional DNA nanostructure assembly, NF assembly is independent of template sequences, avoiding the otherwise complicated design of DNA building blocks assembled into nanostructures by base‐pairing. The NFs were uniform and exhibited high fluorescence intensity and excellent photostability. Combined with the ability of traceable targeted drug delivery, these colorful DNA NFs provide a novel system for applications in multiplex fluorescent cellular imaging, effective screening of drugs, and therapeutic protocol development.     

Endless: A Purine‐Binding RNA Motif that Can Be Expressed in Cells

It is becoming increasingly clear that nature uses RNAs extensively for regulating vital functions of the cell, and short sequences are frequently used to suppress gene expression. However, controlling the concentration of small molecules intracellularly through designed RNA sequences that fold into ligand‐binding structures is difficult. The development of “endless”, a triplex‐based folding motif that can be expressed in mammalian cells and binds the second messenger 3′,5′‐cyclic guanosine monophosphate (cGMP), is described. In vitro, DNA or RNA versions of endless show low micromolar to nanomolar dissociation constants for cGMP. To test its functionality in vivo, four endless RNA motifs arranged in tandem were co‐expressed with a fluorescent cGMP sensor protein in murine vascular smooth muscle cells. Nitric oxide induced endogenous cGMP signals were suppressed in endless‐expressing cells compared to cells expressing a control motif, which suggests that endless can act as a genetically encoded cGMP sink to modulate signal transduction in cells.     

基于磁珠-适配体的高效液相色谱-串联质谱法检测食品中的黄曲霉毒素B1

本文构建了特异性识别黄曲霉毒素B₁(AFB₁)的磁珠-适配体,并与高效液相色谱-串联质谱联用(LC-MS/MS),建立食品中AFB₁的定量检测方法。 利用碳二亚胺盐酸盐(EDC)活化法,将羧基磁珠进行活化。 活化后的羧基磁珠与5'端氨基修饰的适配体进行孵育结合,通过酰胺反应将适配体共价连接在羧基磁珠表面,固定在磁珠表面的适配体作为捕捉探针将样品提取液中的AFB₁分离,通过LC-MS/MS对AFB₁进行定性和定量分析。 检测结果表明:AFB₁在浓度0.25～25 ng/mL呈良好的线性关系,相关系数R²=0.999,定量检出限为0.25 ng/mL,回收率达到80.3%～92.5%,相对标准偏差(RSD)低于8%。 该方法操作简单、快速便捷、可痕量地检测AFB₁,所制备的磁珠-适配体可重复利用,为定量检测AFB₁提供了另一种技术支持。     

The Diversity of a Polyclonal FluCell-SELEX Library Outperforms Individual Aptamers as Emerging Diagnostic Tools for the Identification of Carbapenem Resistant Pseudomonas aeruginosa

Textbook procedures require the use of individual aptamers enriched in SELEX libraries which are subsequently chemically synthesized after their biochemical characterization. Here we show that this reduction of the available sequence space of large libraries and thus the diversity of binding molecules reduces the labelling efficiency and fidelity of selected single aptamers towards different strains of the human pathogen Pseudomonas aeruginosa compared to a polyclonal aptamer library enriched by a whole-cell-SELEX involving fluorescent aptamers. The library outperformed single aptamers in reliable and specific targeting of different clinically relevant strains, allowed to inhibit virulence associated cellular functions and identification of bound cell surface targets by aptamer based affinity purification and mass spectrometry. The stunning ease of this FluCell-SELEX and the convincing performance of the P. aeruginosa specific library may pave the way towards generally new and efficient diagnostic techniques based on polyclonal aptamer libraries not only in clinical microbiology.