Single-Cell Biophysics: Measurement, Modulation, and Modeling

Single-Cell Biophysics: Measurement, Modulation, and Modeling

Poster Abstracts

91-POS

Board 46

Highly Specific mRNA Transcript Quantification in Budding Yeast via Strand

Displacement Induced Hybridization Chain Reaction

Gable Wadsworth

, Harold Kim.

Georgia Institute of Technology, Atlanta, GA, USA.

Quantitative determination of mRNA copy number distribution is essential for understanding

phenotypic variability of single cells. Long mRNA transcripts (> 500 nt) can be detected using

single-molecule Fluorescence In Situ Hybridization (FISH), where the target mRNA is

hybridized with a large number of fluorescently labeled DNA probes. One downside to this

method is the inability to interrogate short targets and short polymorphisms in sequence. We

demonstrate a novel mRNA detection protocol in budding yeast based on strand displacement

induced hybridization chain reaction (HCR). In this method we target a short 10 nucleotide

sequence with an HCR amplifier which is comprised of a pair of DNA hairpins with an exposed

input toehold and a sequestered output toehold. When the target mRNA is present,

polymerization of the HCR amplifier is induced. Sensitive discrimination of the input mRNA

sequence is demonstrated by placing mismatches in the stem of the hairpin. To our knowledge,

this is the first demonstration of using an HCR amplifier to detect mRNA in yeast. Our method

represents an efficient means to obtain quantification of either short mRNA transcripts or short

polymorphisms in mRNA sequence.