单细胞mRNA成像：ligation-bHCR策略实现高灵敏特异突变检测

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"2017ac-单细胞mRNA成像"是一项前沿的科研成果，它专注于提升在分子医学和临床诊断领域中的基因表达可视化技术。研究者们开发了一种创新的荧光原位杂交(FISH)策略，其核心是基于新的分支型杂交链反应（bHCR）信号放大机制。bHCR是一种高度敏感且特异性的技术，相较于传统的FISH方法，它能实现更高效信号放大和更高的检测灵敏度。这项策略突破了以往的技术局限，首次将杂交链反应应用于FISH检测突变，实现了在体外实验中对单核苷酸变异的精确区分。通过使用连接酶介导的区分机制，ligation-bHCR能够确保高特异性，即使在单个mRNA分子水平也能准确识别并区分不同类型的突变。其产生的高度分支聚合物产物，进一步增强了信号的强度和检测效果。在实际成像分析中，ligation-bHCR技术能够生成明亮的斑点信号，这些斑点可以准确地定位单个mRNA分子，使得不同颜色的斑点信号能够作为个体mRNA基因分型的可靠依据。此外，这项策略展现出在单细胞水平上定量检测mRNA表达的巨大潜力，这对于个性化医疗和精准诊断具有重要意义。这种新型的ligation-bHCR FISH策略不仅提供了前所未有的敏感性和特异性，还为开发适用于多种诊断和研究应用的高精度FISH方法开辟了新的可能性。它革新了我们在分子层面理解基因表达变化的方式，对于推动生物医学领域的研究和临床实践具有深远的影响。"

In Situ Imaging of Individual mRNA Mutation in Single Cells Using

Ligation-Mediated Branched Hybridization Chain Reaction (Ligation-

bHCR)

Ying Tang, Xiao-Li Zhang, Li-Juan Tang, Ru-Qin Yu, and Jian-Hui Jiang*

State Key Laboratory of Chemeo/Bio-Sensing and Chemometrics, Institute of Chemical Biology and Nanomedicine, College of

Chemistry and Chemical Engineering, Hunan University, Changsha 410082, P. R. China

Supporting Information

ABSTRACT: Ultrasensitive and speciﬁc in situ imaging of

gene expression is essential for molecular medicine and clinical

theranostics. We develop a novel ﬂuorescence in situ

hybridization (FISH) strategy based on a new branched

hybridization c hain reaction (bHCR) for eﬃcient signal

ampliﬁcation in the FISH assay and a ligase-mediated

discrimination for speciﬁc mutation detection. To our

knowledge, this is the ﬁrst time that HCR has been realized

for mutation detection in the FISH assay. In vitro assay shows

that the ligation-bHCR strategy aﬀords high speciﬁcity in

discriminating single-nucleotide variation in mRNA, and it

generates a highly branched polymeric product that confers more eﬃcient ampliﬁcation or better sensitivity than HCR. Imaging

analysis reveals that ligation-bHCR generates highly bright spot-like signals for localization of individual mRNA molecules, and

spot signals of diﬀerent colors are highly speciﬁc in genotyping point mutation of individual mRNA. Moreover, this strategy is

shown to have the potential for quantitative imaging of the expression of mRNA at the single-cell level. Therefore, this strategy

may provide a new promising paradigm in developing highly sensitive and speciﬁc FISH methods for various diagnostic and

research applications.

etection of gene expression in signal transduction

pathways is essential for be tter unde rstanding of

mechanisms underlying various diseases and for development

of precise theranostic design s.

Curr ent population-based

assays, which are performed on crude tissue extracts to analyze

the content from many cells, typically fail to provide gene

signatures in rare cells.

Increasing knowledge about cellular

and tissue heterogeneity in complex diseases such as cancer has

highlighted the demand for gene detection technologies at the

single-cell level, preferentially with single-molecule sensitivity.

3,4

Fluorescence in situ hybridization (FISH) is a pivotal tool for

genetic analysis at single-cell resolution, which allows direct

mapping of gene expression in a morphological context in the

subcellular and tissue scale.

However, current methods of

FISH still suﬀers from limitations of low signal intensity or

sensitivity and compromised speciﬁcity.

These limitations

create hurdles for FISH in meeting the needs critical for various

diagnostic and research applications, such as detecting genes

such as mRNA with very low copy numbers and resolving a

sequence of high similarity such as point mutations.

Development of the next-generation FISH methodologies

that enable ultrasensitive and highly selective in situ imaging

of mRNA point mutation at single-molecule resolution remains

a great challenge.

Rolling-circle ampliﬁcation (RCA)-based

7,8

FISH (RCA-

FISH) is a useful strategy for highly sensitive and selective

genetic analysis of DNA,

mRNA,

and miRNA.

11,12

The

RCA-FISH strategy is able to provide information about the

localization of nucleic acid targets at the single-molecule level

with single-molecule sensitivity. Recent eﬀort in RCA assay has

been successfully implemented for in situ detection of mRNA

point mutations

and protein complexes.

Despite the

success, RCA-FISH typically shows a limited detection

eﬃciency because in situ ligation of circular probes may be

subjected to topological constraints and lower eﬃciency than

that for linear probes, and ampliﬁcation reaction may be

blocked by nucleic acid targets or their binding proteins.

15,16

address this diﬃculty, hybridization chain reaction (HCR), an

emerging nonenzymatic nucleic acid circuits for signal

ampliﬁcation and detection,

17,18

has been recently developed

for multiplexed FISH of mRNA.

Without the need for

polymerase-based ampliﬁcation, HCR-F ISH exh ibit s deep

penetration, high contrast, and sharp localization. However,

because stringent hybridization conditions for FISH reduce the

energetic driving force for HCR polymerization and thus

decrease the sensitivity, complicated optimization of HCR

probes has to used to increase the signal-to-background ratio in

Received: November 3, 2016

Accepted: February 20, 2017

Published: February 20, 2017

Article

pubs.acs.org/ac

Anal. Chem. 2017, 89, 3445−3451

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