Synthetic DNA Aptamers to Detect Protein Molecular Variants in a High‐Throughput Fluorescence Quenching Assay

适体 猝灭(荧光) DNA 荧光团 荧光 化学 分子探针 高通量筛选 分子结合 微量滴定板 靶蛋白 生物化学 分子生物学 生物物理学 生物 计算生物学 基因 分子 有机化学 物理 量子力学
作者
Xiaohong Fang,Arup Sen,Marie C. Vicéns,Weihong Tan
出处
期刊:ChemBioChem [Wiley]
卷期号:4 (9): 829-834 被引量:166
标识
DOI:10.1002/cbic.200300615
摘要

Abstract 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.

科研通智能强力驱动
Strongly Powered by AbleSci AI
更新
大幅提高文件上传限制,最高150M (2024-4-1)

科研通是完全免费的文献互助平台,具备全网最快的应助速度,最高的求助完成率。 对每一个文献求助,科研通都将尽心尽力,给求助人一个满意的交代。
实时播报
慕青应助有风的晴天采纳,获得10
2秒前
在水一方应助科研通管家采纳,获得10
4秒前
火山应助科研通管家采纳,获得10
4秒前
tuanheqi应助科研通管家采纳,获得50
4秒前
4秒前
CodeCraft应助科研通管家采纳,获得30
4秒前
Leif给倪倪的求助进行了留言
5秒前
6秒前
传奇3应助yuyu采纳,获得10
6秒前
7秒前
GRG完成签到 ,获得积分10
9秒前
maguakai发布了新的文献求助10
9秒前
小马甲应助jz采纳,获得10
10秒前
12秒前
12秒前
13秒前
医学生发布了新的文献求助10
16秒前
第九个现代化完成签到 ,获得积分10
19秒前
爱静静应助Q123ba叭采纳,获得10
20秒前
kaolatong完成签到,获得积分10
21秒前
Hello应助湘江雨采纳,获得10
23秒前
23秒前
shidandan完成签到 ,获得积分10
24秒前
医学生完成签到,获得积分10
27秒前
27秒前
kingwill应助jam采纳,获得20
28秒前
canavalia完成签到,获得积分10
29秒前
希望天下0贩的0应助replay采纳,获得10
29秒前
guozizi应助萧水白采纳,获得100
29秒前
34秒前
彭于晏应助北城采纳,获得10
36秒前
Hyperme完成签到,获得积分10
36秒前
37秒前
39秒前
科研通AI2S应助自由的尔蓉采纳,获得10
42秒前
44秒前
47秒前
48秒前
领导范儿应助aikeyan采纳,获得10
48秒前
华仔应助sun采纳,获得30
48秒前
高分求助中
Production Logging: Theoretical and Interpretive Elements 2000
Very-high-order BVD Schemes Using β-variable THINC Method 1200
中国荞麦品种志 1000
BIOLOGY OF NON-CHORDATES 1000
Autoregulatory progressive resistance exercise: linear versus a velocity-based flexible model 550
The Collected Works of Jeremy Bentham: Rights, Representation, and Reform: Nonsense upon Stilts and Other Writings on the French Revolution 320
Discourse, Identities and Genres in Corporate Communication 300
热门求助领域 (近24小时)
化学 医学 生物 材料科学 工程类 有机化学 生物化学 物理 内科学 纳米技术 计算机科学 化学工程 复合材料 基因 遗传学 物理化学 催化作用 细胞生物学 免疫学 冶金
热门帖子
关注 科研通微信公众号,转发送积分 3359852
求助须知:如何正确求助?哪些是违规求助? 2982410
关于积分的说明 8703731
捐赠科研通 2664107
什么是DOI,文献DOI怎么找? 1458854
科研通“疑难数据库(出版商)”最低求助积分说明 675293
邀请新用户注册赠送积分活动 666410