title: Efficient in situ detection of mRNAs using the Chlorella virus DNA ligase for padlock probe ligation.
abstract: Padlock probes are single-stranded DNA molecules that are circularized upon hybridization to their target sequence by a DNA ligase. In the following, the circulated padlock probes are amplified and detected with fluorescently labeled probes complementary to the amplification product. The hallmark of padlock probe assays is a high detection specificity gained by the ligation reaction. Concomitantly, the ligation reaction is the largest drawback for a quantitative in situ detection of mRNAs due to the low affinities of common DNA or RNA ligases to RNA-DNA duplex strands. Therefore, current protocols require that mRNAs be reverse transcribed to DNA before detection with padlock probes. Recently, it was found that the DNA ligase from Paramecium bursaria Chlorella virus 1 (PBCV-1) is able to efficiently ligate RNA-splinted DNA. Hence, we designed a padlock probe assay for direct in situ detection of mRNAs using the PBCV-1 DNA ligase. Experimental single-cell data were used to optimize and characterize the efficiency of mRNA detection with padlock probes. Our results demonstrate that the PBCV-1 DNA ligase overcomes the efficiency limitation of current protocols for direct in situ mRNA detection, making the PBCV-1 DNA ligase an attractive tool to simplify in situ ligation sequencing applications.
authors.affiliation: Microfluidic and Biological Engineering, Department of Microsystems Engineering-IMTEK, University of Freiburg, 79110 Freiburg, Germany.|Centre for Biological Signalling Studies-BIOSS, University of Freiburg, 79104 Freiburg, Germany.
authors.forename: Nils
authors.initials: N
authors.lastname: Schneider
authors.affiliation: Microfluidic and Biological Engineering, Department of Microsystems Engineering-IMTEK, University of Freiburg, 79110 Freiburg, Germany.|Centre for Biological Signalling Studies-BIOSS, University of Freiburg, 79104 Freiburg, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Meier
mesh_terms: D000199:Actins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D020029:Base Pairing; D011088:DNA Ligases / Q000737:chemistry*; D015342:DNA Probes / Q000737:chemistry*; D004277:DNA, Single-Stranded / Q000737:chemistry*; D005404:Fixatives / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D059010:Single-Cell Analysis / Q000379:methods; D016707:Tissue Fixation; D014764:Viral Proteins / Q000737:chemistry*
chemical_list: D000199:Actins; D015342:DNA Probes; D004277:DNA, Single-Stranded; D005404:Fixatives; D005456:Fluorescent Dyes; D012333:RNA, Messenger; D014764:Viral Proteins; D005557:Formaldehyde; C110456:Chlorella virus DNA ligase; D011088:DNA Ligases
keywords: DNA padlock probes; in situ ligation; mRNA quantification