An RNA Sensor for Detection of Circulating Tumor Cells

用于检测循环肿瘤细胞的 RNA 传感器

• 批准号: 7337060
• 负责人: GARY A CLAWSON
• 金额: $ 28.45万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-15 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7337060
• 关键词: Air; Antisense Oligonucleotides; Arts; Binding; Biological Assay; Biosensor; Breast Melanoma; Clinical; Clinical Research; Compatible; Complex; Condition; Data; Deposition; Detection; Development; Devices; Disease; Event; Frequencies; Funding; Glass; Gold; Libraries; Liquid substance; MART-1 Tumor Antigen; Malignant Neoplasms; Malignant neoplasm of prostate; Measurement; Measures; Melan-A protein; Methods; Nucleic Acids; Optics; Population; Preparation; Process; Prostatic; Protocols documentation; RNA; RNA Binding; Range; Reading; Research Personnel; Sampling; Silicon; Site; Slide; Specificity; System; Technology; Testing; base; cancer type; cantilever; cell type; design; high throughput screening; malignant breast neoplasm; melanoma; nanoprobe; nanowire; neoplastic cell; particle; pathogen; programs; research study; sensor; validation studies; voltage

This proposal seeks to develop an RNA Sensor to be employed for detection of circulating tumor cells. RNA detection is based upon an hybridization "sandwich". Two target RNAs have been chosen for clinically important cancers (prostate, breast, and melanoma), and library selection protocols will be utilized to identify/optimize accessible sites for antisense oligonucleotide (ASO) binding. Silicon nanowires will then be covalently derivatized with ASO to a library-selected site (ASO-,) in the target RNA. The ASOi nanowires will then be deposited by fluidic deposition onto chips, and integrated into the underlying CMOS circuitry. Target RNA will be purified from cellular preparations, and will then be hybridized to the ASd-nanowires. An ASO2, targeted to a 2nd library-selected site, will be covalently attached to 12 nm gold particles (ASO2-nanoprobe). Binding of the ASO2-nanoprobe to the target RNA-ASOi-nanowire complexes will induce a resonance frequency shift in the nanowires, which is greatly amplified by the mass of the gold particle. This resonance frequency shift (RXA)will be detected by direct electrical read-out, with voltage (quantitatively) related to binding events (RX,A) will initially be detected optically). We have successfully measured RX of 300 nm silicon nanowires (with high Quality-Factors) under ambient conditions. Theoretical calculations predict very good Quality-Factors for silicon nanowires in H20, and detection of single binding events should be achievable. Preliminary data related to all aspects of RNA Sensor development have been obtained. These include: library selection of target sites in prostatic DD3 RNA, sandwich hybridization specificity "off-chip" synthesis and derivatization of nanowires, R>. measurements with nanowires, and fluidic deposition of nanowires on chips. After basic developmental steps are completed, experiments will include quantitative determination of target RNAs using the detection device compared to QPCR amplification. The Specific Aims for this funding period are designed to develop an RNA Sensor appropriate for subsequent use in clinical validation studies for circulating tumor cells. Successful development of this RNA Sensor would provide a major advantage over PCR-based assays, and could form the basis for high-throughput screening tests for simultaneous detection of many different circulating tumor cell types.

这项建议旨在开发一种RNA传感器，用于检测循环中的肿瘤细胞。 RNA检测是基于杂交“三明治”进行的。已经选择了两个靶向RNA用于临床 重要的癌症(前列腺癌、乳腺癌和黑色素瘤)和文库选择方案将用于 确定/优化反义寡核苷酸(ASO)结合的可访问位置。然后，硅纳米线将被 与ASO共价衍生到目标RNA中的文库选择的位置(ASO-，)。ASOI纳米线将 然后通过流控沉积将其沉积到芯片上，并集成到底层的CMOS电路中。目标 RNA将从细胞制剂中提纯，然后与ASD纳米线杂交。一个ASO2， 靶向第二个文库选择的位置，将共价连接到12 nm的金颗粒(ASO2-纳米探针)。 ASO2-纳米探针与靶RNA-ASOI-纳米线复合体的结合将引起共振 纳米线中的频移，它被金粒子的质量极大地放大。这种共鸣 频移(RXA)将通过直接电读出进行检测，电压(定量)与 结合事件(RX、A)最初将被光学检测)。我们已经成功地测量了300 nm的Rx 环境条件下的硅纳米线(具有高品质因数)。理论计算预测非常 H20中的硅纳米线的良好品质因数，并且应该检测到单个结合事件 是可以实现的。 与RNA传感器发展的各个方面有关的初步数据已经获得。这些措施包括： 前列腺靶基因DD3RNA的文库选择、夹心杂交特异性“芯片外”合成 和纳米线的衍生化，R&gt；。用纳米线进行测量，并对纳米线进行流体沉积 薯片。在基本开发步骤完成后，实验将包括定量测定 利用该检测装置对靶RNA进行检测，并与QPCR扩增进行比较。这笔资金的具体目标是 Period的目的是开发一种适合于随后用于临床验证研究的RNA传感器 用于循环中的肿瘤细胞。这种RNA传感器的成功开发将提供一个重大优势 超过了基于聚合酶链式反应的检测，并可构成同时进行高通量筛查试验的基础。 检测多种不同的循环肿瘤细胞类型。

项目成果

Primer-free aptamer selection using a random DNA library.

使用随机 DNA 文库进行无引物适体选择。

• DOI: 10.1007/978-1-60761-657-3_24
• 发表时间: 2010
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Pan,Weihua;Clawson,GaryA
• 通讯作者: Clawson,GaryA

Nanoresonator chip-based RNA sensor strategy for detection of circulating tumor cells: response using PCA3 as a prostate cancer marker.

• DOI: 10.1016/j.nano.2011.11.009
• 发表时间: 2012-08
• 期刊: NANOMEDICINE-NANOTECHNOLOGY BIOLOGY AND MEDICINE
• 影响因子: 5.4
• 作者: Sioss, James A.;Bhiladvala, Rustom B.;Pan, Weihua;Li, Mingwei;Patrick, Susan;Xin, Ping;Dean, Stacey L.;Keating, Christine D.;Mayer, Theresa S.;Clawson, Gary A.
• 通讯作者: Clawson, Gary A.

The shorter the better: reducing fixed primer regions of oligonucleotide libraries for aptamer selection.

• DOI: 10.3390/molecules14041353
• 发表时间: 2009-03-27
• 期刊: Molecules (Basel, Switzerland)
• 作者: Pan W;Clawson GA
• 通讯作者: Clawson GA

MicroRNAs align with accessible sites in target mRNAs.

MicroRNA 与目标 mRNA 中的可接近位点对齐。

• DOI: 10.1002/jcb.22428
• 发表时间: 2010
• 期刊: Journal of cellular biochemistry
• 影响因子: 4
• 作者: Pan,Weihua;Xin,Ping;Clawson,GaryA
• 通讯作者: Clawson,GaryA