Single-molecule counting of circular RNAs using phage nanoparticles as surrogates
使用噬菌体纳米颗粒作为替代物对环状 RNA 进行单分子计数
基本信息
- 批准号:10092159
- 负责人:
- 金额:$ 22.79万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2020
- 资助国家:美国
- 起止时间:2020-02-01 至 2022-05-06
- 项目状态:已结题
- 来源:
- 关键词:AffinityBacteriaBacteriophage T7BacteriophagesBindingBiological AssayBiological MarkersBiomedical EngineeringBiosensing TechniquesBody FluidsBreast Cancer CellBreast Cancer PatientBuffersCapsidCellsChemicalsClinicCodeColorComplementary DNAComplexDetectionEngineeringEyeFiberGeneticGenetic EngineeringGoalsHourHumanIn VitroLettersMagnetismMammary NeoplasmsMammographyMethodsModificationNanotechnologyNucleic AcidsOligonucleotidesPeptidesPhosphate BufferPolymerase Chain ReactionProteinsRNAReproducibilityResolutionReverse TranscriptionSalineSamplingSensitivity and SpecificitySeriesSerumSodium ChlorideSpecificityStructureTailTechniquesTestingTimeTissuesTumor TissueUntranslated RNAVirusVirus-like particleVisualWaterWomanaptameraqueousbasebreast cancer diagnosiscancer biomarkerscancer diagnosiscancer therapycircular RNAcostdesigndetection limitdigitalearly detection biomarkersmalignant breast neoplasmmillimeternanobiomaterialnanomaterialsnanoparticlenoveloverexpressionparticleprotein biomarkersred fluorescent proteinsingle molecule
项目摘要
Single-molecule counting of circular RNAs using phage nanoparticles as surrogates
Abstract
Circular RNA (circRNA) have been considered as promising breast cancer biomarkers, but the current qRT-PCR
and digital PCR techniques for quantifying them have some inherent limitations. T7 phage, a human-safe virus
nanoparticle specifically infecting bacteria, can be plated to infect bacteria in a plaque-forming assay to form
millimeter-scale plaques in a one to one format and at a single-particle resolution within 3 hours. Inspired from
this, we propose to develop a phage plaque counting (PPC) strategy that hires bioengineered fluorescent T7
phage as a surrogate to establish a one-to-one correspondence among target circRNA, phage nanoparticles,
and eye-visible phage-developed plaques, enabling us to simultaneously quantify multiple circRNA biomarkers
in a single test by simply counting the corresponding plaques. Briefly, a fluorescent phage probe with an
oligonucleotide (ONT-1) capable of capturing one unique segment of a target circRNA, and a magnetic
microparticle (MMP) probe with an ONT-2 capable of capturing another unique segment of the same target, co-
capture the target to form a sandwich complex where phages and target molecules are equimolar. Then the
phages are released and plated to develop fluorescent plaques in a one to one format, thus counting the plaques
at the single-particle level leads to visualized quantification of the target circRNA at a single-molecule level. A
fluorescent protein on the capsid makes the corresponding plaque fluoresce a unique color and enables the
simultaneous single-particle quantification of fluorescent plaques (in the same Petri dish) with each color coding
one target (i.e., by displaying green and red fluorescent protein for two corresponding targets). We hypothesize
that our PPC strategy with optimized conditions can simultaneously quantify a panel of two circRNAs as breast
cancer biomarkers in human serum with high sensitivity, specificity, and reproducibility. Aim 1: Establish and
optimize PPC strategy for quantifying single and multiple circRNA breast cancer biomarkers. We will
produce and purify the target circRNAs (circ_0001785 and circ_100219) by the overexpression method and use
the PPC method to quantify them with a series of dilutions in water. We will optimize the PPC and identify its
detection limit. Aim 2: Validate the PPC strategy for simultaneously quantifying multiple circRNA
biomarkers in breast cancer cells, tissues, and human serum. We will first use MMPs that can capture the
target circRNAs to magnetically remove the pre-existing target circRNAs from the commercial human serum,
which is then used to make serum samples with known concentrations of target circRNAs. Then we will employ
and optimize the PPC to quantify the target circRNAs in the serum. We will also isolate total RNAs from breast
cancer cells and in vitro breast tumor tissues to form aqueous RNA solutions by a commercial RNA isolation kit.
We will then use the PPC to quantify the target circRNAs in the resultant RNA solutions and the commercial total
RNA solutions isolated from tumor tissues of breast cancer patients. This project will lead to a new visualized
single-molecule technique for quantifying circRNA biomarkers for breast cancer diagnosis.
以噬菌体纳米颗粒为替代物的环状rna单分子计数
项目成果
期刊论文数量(0)
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