Synthetic DNA aptamers to detect protein molecular variants in a high-throughput fluorescence quenching assay

Real-time protein detection in homogeneous solutions is necessary in many biotechnology and biomedical studies. The recent development of molecular aptamers, combined with fluorescence techniques, may provide an easy and efficient approach to protein elucidation. This report describes the development of a fluorescence-based assay with synthetic DNA aptamers that can detect and distinguish molecular variants of proteins in biological samples in a high-throughput process. We used an aptamer with high affinity for the B chain of platelet-derived growth factor (PDGF), labeled it with a fluorophore and a quencher at the two termini, and measured fluorescence quenching by PDGF. The specific quenching can be used to detect PDGF at picomolar concentrations even in the presence of serum and other cell-derived proteins in cell culture media. This is the first successful application of a synthetic aptamer for the detection of tumor-related proteins directly from the tumor cells. We also show that three highly related molecular variants of PDGF (AA, AB, and BB dimers) can be distinguished from one another in this single-step assay, which can be readily adapted to a microtiter plate assay for high-throughput analysis. The use of fluorescence quenching as a measure of binding between the DNA probe and the target protein eliminates potential false signals that may arise in traditional fluorescence enhancement assays as a result of degradation of the DNA aptamer by contaminating nucleases in biological specimens. This assay is applicable to proteins that are not naturally DNA binding. The excellent specificity, ultrahigh sensitivity, and simplicity of this one-step assay addresses a growing need for high-throughput methods that detect changes in the expression of gene products and their variants in cell cultures and biological specimens.     

Investigation of molecular beacon aptamer-based bioassay for platelet-derived growth factor detection

This report describes studies on the use of a molecular-beacon aptamer (MBA) as a synthetic high-affinity DNA probe that exhibits fluorescence resonance energy transfer (FRET) in response to a specific protein biomarker, platelet-derived growth factor (PDGF). As a step toward the application of the MBA in a fluorescence-based assay for biological specimens, we examined the influence of certain physical and chemical parameters of incubation that would affect DNA conformation and DNA-backbone modification, and thus improve nuclease resistance. This bioassay is compatible with pH, temperature, and monovalent cation levels typically encountered in biological samples, and phosphorothioate backbone-modified MBA is able to exhibit specific FRET. With minimal sample processing and without assay optimization, the MBA is able to detect as little as 10 ng PDGF per mug of serum proteins from cell-culture media. We also show that different sets of known fluorophore-quencher pairs can be successfully used in the MBA for sensitive detection of the PDGF target. It should, therefore, be possible to develop multiplex bioassays that monitor either quenching or enhancement for the simultaneous detection of several biomarkers by using MBAs created from high-affinity DNA ligands for the desired protein targets. Interestingly, we observed that, with a DNA ligand with multiple binding sites for a standard multimeric protein target, the FRET bioassay could be accomplished by using a mixture of two individually labeled DNAs-one carrying the fluorophore and the other with the matching quencher. This observation has significant implications in the future design of more selective DNA-based FRET bioassays that use more than one ligand for the same protein target.     

吖啶类荧光探针与蛋白质相互作用的研究

文章研究3种新型吖啶类荧光探针(N-(2-二甲氨基)乙基-9-氯吖啶-4-甲酰胺(NCAF)、9-[(N-2-二甲氨乙基)吖啶-4-甲酰胺]-α-丙氨酸(NAFA)和4,9-二[N-(2-二甲氨基)乙基]-9-吖啶胺-4-甲酰胺(DNAF))与牛血清白蛋白(BSA)的相互结合作用机理。分别对3种吖啶类探针自聚集情况、与牛血清白蛋白结合常数KA和结合位点数n、热力学参数H、G以及S、能量转移效率E和结合距离R0进行比较,并对实验数据进行分析。研究了3种吖啶类探针对蛋白质内源荧光猝灭的猝灭机制和主要作用力类型,为进一步研究新的生物探针及其在生物大分子识别分析应用提供了一定的实验和理论支持。     

L-半胱氨酸与牛血清白蛋白相互作用的荧光光谱分析

运用荧光猝灭光谱、同步荧光光谱探讨了L-半胱氨酸（L-Cys）与牛血清白蛋白（BSA）的相互作用,并计算了猝灭常数、结合常数、结合位点数以及3个热力学参数△H、△G和△S。结果表明,L-Cys使BSA的内源荧光发生猝灭,BSA的发射峰从350nm蓝移到347．5nm,荧光猝灭机制为动态猝灭;L-Cys与BSA之间的作用力主要为疏水作用力;L-Cys对BSA结构的微环境有一定的影响。     

苯甲酸与牛血清白蛋白相互作用的荧光光谱研究

采用荧光光谱、三维荧光光谱、同步荧光光谱研究了苯甲酸与牛血清白蛋白(BSA)分子之间的相互作用机制、特征及苯甲酸对牛血清白蛋白构象的影响。根据不同温度下苯甲酸对牛血清白蛋白的荧光猝灭作用,利用Stem-Volmer和Lineweaver-Burk方程和热力学方程分别处理实验数据,求得它们之间的结合常数K为1.096×103L/mol、结合位点数n为1.12(20℃),ΔH=-31.8 kJ/mol,ΔG=-17.1 kJ/mol,ΔS=-50.2 J.mol-1.K-1。研究发现苯甲酸对BSA内源性荧光的猝灭作用属于静态猝灭,苯甲酸与BSA分子之间的相互作用是一个吉布斯自由能降低的自发过程,两者之间的主要作用力类型为氢键力或范德华力。     

4,9-二[N-(2-二甲氨基)乙基]-9-吖啶胺-4-甲酰胺与蛋白质相互作用

应用水溶性4,9一二[N-(2一二甲氨基)乙基]-9-吖啶胺-4-甲酞胺(DNAF)探针,研究了DNAF与牛血清白蛋白(BSA)的相互结合作用机理.计算出相应的热力学参数△H,△G以及△S;通过荧光猝灭方法研究结合作用,测定出结合位点数n和表观结合常数KA;根据荧光共振能童转移理论求得供体BSA和受体DNAF的距离r;...     

MBNL1-RNA recognition: contributions of MBNL1 sequence and RNA conformation

Muscleblind-like proteins (MBNL) are RNA-binding proteins that bind to the poly(CUG) and poly(CCUG) sequences that are the causative agents of myotonic dystrophy. It has been suggested that as a result of binding to the repeating RNA sequences, MBNL1 is abnormally expressed and translocated, which leads to many of the misregulated events in myotonic dystrophy. In this work, steady-state fluorescence quenching experiments suggest that MBNL1 alters the structure of helical RNA targets upon binding, which may explain the selectivity of MBNL1 for less structured RNA sites. The removal of one pair of zinc fingers greatly impairs the binding affinity of MBNL1, which indicates that the two pairs of zinc fingers might possibly interact with RNA targets cooperatively. Alanine scanning mutagenesis results suggest that the binding energy may be distributed across the protein. Overall, the results presented here suggest that small molecules that stabilize the helical structure of poly(CUG) and poly(CCUG) RNAs will inhibit the formation of complexes with MBNL1.     

荧光光谱法研究核黄素与牛血清白蛋白的相互作用

荧光光谱法研究了注射用核黄素(Riboflavin,Rf)与牛血清白蛋白(BSA)的相互作用.核黄素对BSA具有荧光猝灭作用,其猝灭方式为静态猝灭;在301和317K下用Stem-Volmer方程和热力学方程等处理实验数据,求出了结合常数KA、结合位点数n及热力学参数ΔG、ΔH和ΔS.核黄素与BSA之间的作用力主要为氢...     

Binding Specificity of Recombinant Odorant-Binding Protein Isoforms is Driven by Phosphorylation

Native porcine odorant-binding protein (OBP) bears eleven sites of phosphorylation, which are not always occupied in the molecular population, suggesting that different isoforms could co-exist in animal tissues. As phosphorylation is a dynamic process resulting in temporary conformational changes that regulate the function of target proteins, we investigated the possibility that OBP isoforms could display different binding affinities to biologically relevant ligands. The availability of recombinant proteins is of particular interest for the study of protein/ligand structure-function relationships, but prokaryotic expression systems do not perform eukaryotic post-translational modifications. To investigate the role of phosphorylation in the binding capacities of OBP isoforms, we produced recombinant porcine OBP in two eukaryotic systems, the yeast, Pichia pastoris, and the mammalian CHO cell line. Isoforms were separated by anion exchange HPLC, and their phosphorylation sites were mapped by MALDI-TOF mass spectrometry and compared to those of the native protein. Binding experiments with ligands of biological relevance in the pig, Sus scrofa, were performed by fluorescence spectroscopy on two isoforms of recombinant OBP expressed in the yeast. The two isoforms, differing only by their phosphorylation pattern, displayed different binding properties, suggesting that binding specificity is driven by phosphorylation.     

荧光光谱法研究二氢卟吩e6和锌二氢卟吩e6与牛血清白蛋白的相互作用

应用紫外光谱和荧光光谱法研究二氢卟吩e6(Ce6)和锌二氢卟吩e6(ZnCe6)分别与牛血清白蛋白(BSA)的相互作用。通过紫外光谱、荧光光谱、光照实验,探讨了两种物质与BSA相互作用的荧光猝灭光谱特征、猝灭机理、与BSA结合反应及光照的影响。结果表明,Ce6和ZnCe6都可与BSA发生相互作用,Ce6和ZnCe6浓度的变化会引起Ce6、ZnCe6与BSA之间能量转移的变化,Ce6的猝灭程度要比ZnCe6强,猝灭机制是由静态猝灭引起的。Ce6与BSA的结合能力强于锌二氢卟吩e6,结合位点数均接近1,结合的作用力类型主要为范德华力和氢键作用力。Ce6-BSA和ZnCe6-BSA复合物均有光漂白特性,光漂白机制均为光学修饰型引起的,这种机制有利于提高光动力治疗(PDT)效果。     

一种新型喹唑啉酮衍生物A与牛血清白蛋白作用的研究

应用荧光光谱和紫外可见吸收光谱研究了A与牛血清白蛋白间的结合作用,确定了A对牛血清白蛋白的荧光猝灭过程的猝灭机理。测定了不同温度下该结合反应的结合常数,结合位点数,热力学参数。依据能量转移理论确定了A和蛋白间的结合距离。采用同步荧光技术考察了A对牛血清白蛋白构象的影响,并讨论了A与蛋白的结合模式。     

Conjugated fluorescent polymer sensor for proteolytic activity detection with designed specificity

A portable fluorescence assay for direct endopeptidase activity detection has been developed with the use of a cyclophane‐based conductive conjugated fluorescent polymer and peptide substrates. The substrates, carrying internal quenching amino acid, were designed to be cleaved in a sequence‐specific manner by a protease of interest. Intact substrates were incapable of quenching the fluorescence of the polymer due to steric constraints. Upon specific cleavage, the quencher became exposed and could interact with the ring structure of the fluorescent polymer, disrupting the conjugation and quenching the fluorescence along the polymer chain. The approach was developed using a model Glu‐C endopeptidase from Staphylococcus aureus strain V8, detected in the picomolar to micromolar concentration range. The developed assay was tested for the detection of endopeptidase activity of botulinum toxin. The feasibility study showed that botulinum neurotoxin (BoNT)‐A could be detected down to picomolar concentration, with limit of detection of 5 pg, or 33 amol in 5 µL of sample, and a total assay time under 2 h. The assay exhibited high specificity and no cross‐reactivity with BoNT‐B was detected. Following this proof‐of‐concept work, the assay can be further optimized and expanded to differentiate between various botulinum toxin serotypes in their active proteolytic form, or modified for detection of proteases with other specificities. © 2015 Society of Chemical Industry     

Two-step, PCR-free telomerase detection by using exonuclease III-aided target recycling

We report the sensitive detection of telomerase activity by using exonuclease III-aided target recycling to amplify the signal produced by a chimeric LNA-DNA molecular beacon. We demonstrate the specific detection of as few as 30 telomerase-positive breast cancer cells in a single-measurement fluorescence assay that avoids the problematic PCR and gel analysis of the current "gold-standard" assay.     

A Modular Nanoswitch for Mix‐and‐Detect Protein Assay Based on Binding‐Induced Cascade Dissociation of Kissing Complex

A new modular nanoswitch was described for versatile, rapid (within 1 h), homogeneous, and sensitive protein detection. The system employs two hairpins (HP1 and HP2) that can be reciprocally recognized through the apical loop–loop interaction. HP2 possesses a conformation‐switching stem–loop structure, with appended single‐stranded tails on each end, which can hybridize with the recognition‐element‐conjugated DNA strands to construct a protein‐responsive HP2 scaffold. It works according to a simple mix‐and‐detect assay format, with the first formation of a kissing complex between HP1 and HP2 scaffolds for fluorescence quenching, and then cascade propagation from steric strain through protein binding to the dissociation of the kissing complex for fluorescence recovery. The detection universality of such a modular nanoswitch was demonstrated by using three multivalent proteins, including anti‐digoxigenin (Anti‐Dig) antibody, streptavidin, and thrombin, with detection limits of 0.33, 0.17, and 0.5 nm , respectively.     

汞(Ⅱ)与明胶蛋白质相互作用的荧光光谱研究

利用荧光光谱技术,研究了pH为7.30时Hg2+离子与明胶的相互作用,用不同的公式对实验结果进行对比处理,计算了结合常数(K)和结合位点数(n)。推测了Hg2+离子对明胶荧光的猝灭机制为Hg2+离子浓度较低时主要是动态猝灭,浓度较高时表现为静态猝灭。     

Tau as a Biomarker of Neurodegeneration

Less than 50 years since tau was first isolated from a porcine brain, its detection in femtolitre concentrations in biological fluids is revolutionizing the diagnosis of neurodegenerative diseases. This review highlights the molecular and technological advances that have catapulted tau from obscurity to the forefront of biomarker diagnostics. Comprehensive updates are provided describing the burgeoning clinical applications of tau as a biomarker of neurodegeneration. For the clinician, tau not only enhances diagnostic accuracy, but holds promise as a predictor of clinical progression, phenotype, and response to drug therapy. For patients living with neurodegenerative disorders, characterization of tau dysregulation could provide much-needed clarity to a notoriously murky diagnostic landscape.     

Structural Biology for the Molecular Insight between Aptamers and Target Proteins

Aptamers are promising therapeutic and diagnostic agents for various diseases due to their high affinity and specificity against target proteins. Structural determination in combination with multiple biochemical and biophysical methods could help to explore the interacting mechanism between aptamers and their targets. Regrettably, structural studies for aptamer–target interactions are still the bottleneck in this field, which are facing various difficulties. In this review, we first reviewed the methods for resolving structures of aptamer–protein complexes and for analyzing the interactions between aptamers and target proteins. We summarized the general features of the interacting nucleotides and residues involved in the interactions between aptamers and proteins. Challenges and perspectives in current methodologies were discussed. Approaches for determining the binding affinity between aptamers and target proteins as well as modification strategies for stabilizing the binding affinity of aptamers to target proteins were also reviewed. The review could help to understand how aptamers interact with their targets and how alterations such as chemical modifications in the structures affect the affinity and function of aptamers, which could facilitate the optimization and translation of aptamers-based theranostics.