Sequencing microRNAs, with single-base discrimination, using a nanopore device and an Osmium tag.
使用纳米孔装置和锇标签对 microRNA 进行单碱基区分测序。
基本信息
- 批准号:10076687
- 负责人:
- 金额:$ 27.68万
- 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:2020-09-23 至 2021-08-31
- 项目状态:已结题
- 来源:
- 关键词:AgingBindingBiologicalBiological AssayBiological MarkersBloodCationsChargeClinicalCodeComplementary DNAComputer softwareConsensusConsumptionCytidineDNA-Directed RNA PolymeraseDetectionDevicesDiagnosisDiagnosticDiscriminationDiseaseDropsEnzymesEventExhibitsExplosionFingerprintFloodsFundingFutureGene Expression RegulationGenetic TranscriptionGoalsGrantHandednessHealthHealth Care CostsHealth StatusIndividualIndustryInfrastructureIonsLabelLiquid substanceLiteratureMedicalMicroRNAsNational Human Genome Research InstituteNatureNucleic AcidsNucleotidesOsmiumOsmium TetroxidePatternPeptidesPharmaceutical PreparationsPhasePolyaminesPreparationPreventionPropertyProteinsProtocols documentationPublicationsPurinesPyrimidinesRNARNA BindingRNA SequencesRNA StabilityRNA-Binding ProteinsReadabilityRecurrenceReportingResearchResidual stateSamplingSignal TransductionSmall Business Innovation Research GrantSmall RNASpecificitySpermidineSpermineStressTechnologyTemperatureTestingTimeTransfer RNAUnited States National Institutes of HealthUntranslated RNAUracilUrineWorkarginyllysinebaseconstrictiondiagnostic assayindividual responseinnovative technologiesmiRNA expression profilingminimally invasivenanoporenovelpersonalized medicinepolycationportabilitypreservationpreventremote locationresidencesingle moleculesuccesstranscriptomeusabilityvoltage
项目摘要
PROJECT SUMMARY
The goal of this work is to develop an inexpensive, ultra-fast, minimally-invasive medical test to sequence and
quantitate small RNAs from biological fluids. Small RNAs, shorter than 200 nucleotides (nt) comprise several
groups of non-coding RNAs with preserved regulatory functions. Small RNAs are abundant and surprisingly
stable in blood/urine. Current literature is flooded with studies identifying such small RNAs as reliable
biomarkers for most diseases. These studies suggest that a comprehensive small RNA panel will reflect the
health/aging status of an individual, response to stress, changes in medication, onset and/or progress of
disease. Current small RNA profiling assays are expensive, involve extensive infrastructure, and employ time-
consuming and error-prone sample preparation. These assays also miss or misidentify post-transcriptionally
modified bases (PTM) which are known to influence RNA stability, specificity, and function. On the contrary,
nanopore-based technologies promise inexpensive portable devices, direct RNA profiling including
identification of PTM bases, and fast assay turn-around. Still the only commercially available nanopore-based
device(s) from Oxford Nanopore Technologies (ONT) are not qualified to sequence RNAs shorter than 200
bases. Such limitation prevents sequencing of most, if not all, non-coding RNAs found in biological fluids, and
limits the usability of nanopore technology in minimally-invasive medical diagnostic assays.
We will use commercially available nanopore devices and optimize the testing conditions in order to profile
small RNAs in biological fluids. Yenos Analytical LLC is singled out for such effort, as we have developed
proprietary technology to selectively tag RNAs with a bulky Osmium label. The bulky label slows down the
voltage-driven translocation of these RNA surrogates via a nanopore, and yields a readable ion current vs
time (i-t) signal that serves as the fingerprint of the specific RNA molecule. Recent work funded by a Phase I
SBIR NHGRI grant has also shown that some of the slowest translocations of these tagged RNAs using the
ONT device(s) embody sequencing information. In order to increase the number of the slow events, we will
exploit non-covalent probes that bind RNA. This binding will reduce the effective negative charge of the RNA
molecule, and slowly release it under the influence of the voltage drop in proximity to the nanopore’s entry. As
non-covalent probes, we will evaluate cationic polyamines such as spermidine and spermine, cationic
peptides, and RNA binding proteins. The combination of nucleic acid selective labeling technology with
nanopore-device based detection is novel, and single-handedly carried out by Yenos Analytical LLC. We
propose to optimize this combination of technologies towards profiling small RNAs from biological fluids. Such
a medical assay will directly impact medical testing, prevention, diagnosis, and cure of disease, materialize
the promise of personalized medicine, reduce the ballooning cost of health care, and enable diagnostic
medical tests in remote locations with minimal infrastructure.
项目摘要
这项工作的目标是开发一种廉价,超快速,微创的医学测试,以测序和
从生物液体中定量小RNA。短于200个核苷酸(nt)的小RNA包含几个
具有保留的调节功能的非编码RNA组。小RNA是丰富的,令人惊讶的是
在血液/尿液中稳定。目前的文献中充斥着鉴定这种小RNA是可靠的
大多数疾病的生物标志物。这些研究表明,一个全面的小RNA面板将反映
个体的健康/衰老状态、对压力的反应、药物治疗的变化、
疾病目前的小RNA分析测定是昂贵的,涉及广泛的基础设施,并占用时间-
耗时且容易出错的样品制备。这些检测也会遗漏或错误识别转录后
修饰的碱基(PTM),其已知影响RNA稳定性、特异性和功能。与此相反的是,
基于纳米孔的技术承诺廉价的便携式设备,直接RNA分析,包括
PTM碱基的鉴定和快速检测周转。仍然是唯一的商业上可用的基于纳米孔的
Oxford Nanopore Technologies(ONT)的设备不适合对短于200的RNA进行测序
基地这种限制阻止了对生物流体中发现的大多数(如果不是全部)非编码RNA的测序,并且
限制了纳米孔技术在微创医学诊断测定中的可用性。
我们将使用市售的纳米孔设备,并优化测试条件,以便分析
生物体液中的小RNA。Yenos分析有限责任公司是挑选出来的这种努力,因为我们已经开发
专利技术,选择性地标记RNA与庞大的锇标签。笨重的标签减慢了
这些RNA替代物通过纳米孔的电压驱动易位,并产生可读的离子电流与
时间(i-t)信号,其用作特定RNA分子的指纹。最近的工作由第一阶段资助
SBIR NHGRI基金还表明,这些标记RNA的一些最慢的易位使用了
ONT设备包含定序信息。为了增加慢事件的数量,我们将
利用结合RNA的非共价探针。这种结合将减少RNA的有效负电荷
分子,并在接近纳米孔入口的电压降的影响下缓慢释放它。作为
非共价探针,我们将评估阳离子多胺,如亚精胺和精胺,阳离子
肽和RNA结合蛋白。核酸选择性标记技术与
基于纳米孔装置的检测是新颖的,并且由YenosAnalytical LLC单独进行。我们
我们建议优化这种技术组合,以分析来自生物液体的小RNA。等
医学分析将直接影响医学检测、预防、诊断和治疗疾病,
个性化医疗的承诺,降低医疗保健的成本膨胀,并使诊断
在偏远地区进行医疗测试,基础设施很少。
项目成果
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