Biology subjects

Pierce, N. A.

Publications and source records attributed to Pierce, N. A..

3 recordsLinked to original sources

bioRxiv2018

Signal Transduction in Human Cell Lysate via Dynamic RNA Nanotechnology

Dynamic RNA nanotechnology with small conditional RNAs (scRNAs) offers a promising conceptual approach to introducing synthetic regulatory links into endogenous biological circuits. Here, we use human cell lysate containing functional Dicer and RNases as a testbed for engineering scRNAs for conditional RNA interference (RNAi). scRNAs perform signal transduction via conditional shape change: detection of a subsequence of mRNA input X triggers formation of a Dicer substrate that is processed to yield siRNA output anti-Y targeting independent mRNA Y for destruction. Automated sequence design is performed using the reaction pathway designer within NUPACK to encode this conditional hybridization cascade into the scRNA sequence subject to the sequence constraints imposed by X and Y. Because it is difficult for secondary structure models to predict which subsequences of mRNA input X will be accessible for detection, here we develop the RNAhyb method to experimentally determine accessible windows within the mRNA that are provided to the designer as sequence constraints. We demonstrate the programmability of scRNA regulators by engineering scRNAs for transducing in both directions between two full-length mRNAs X and Y, corresponding to either the forward molecular logic \"if X then not Y\" (X [boxvl] Y) or the reverse molecular logic \"if Y then not X\" (Y [boxvl] X). In human cell lysate, we observe a strong OFF/ON conditional response with low crosstalk, corresponding to a {approx}20-fold increase in production of the siRNA output in response to the cognate vs non-cognate full-length mRNA input. Because diverse biological pathways interact with RNA, scRNAs that transduce between detection of endogenous RNA inputs and production of biologically active RNA outputs hold great promise as a synthetic regulatory paradigm.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=63 SRC=\"FIGDIR/small/439273_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (10K):\norg.highwire.dtl.DTLVardef@fb2a0org.highwire.dtl.DTLVardef@98517dorg.highwire.dtl.DTLVardef@e0fe8org.highwire.dtl.DTLVardef@1364f8d_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2018

Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust

In situ hybridization based on the mechanism of hybridization chain reaction (HCR) has addressed multi-decade challenges to imaging mRNA expression in diverse organisms, offering a unique combination of multiplexing, quantitation, sensitivity, resolution, and versatility. Here, with third-generation in situ HCR, we augment these capabilities using probes and amplifiers that combine to provide automatic background suppression throughout the protocol, ensuring that even if reagents bind non-specifically within the sample they will not generate amplified background. Automatic background suppression dramatically enhances performance and robustness, combining the benefits of higher signal-to-background with the convenience of using unoptimized probe sets for new targets and organisms. In situ HCR v3.0 enables multiplexed quantitative mRNA imaging with subcellular resolution in the anatomical context of whole-mount vertebrate embryos, multiplexed quantitative mRNA flow cytometry for high-throughput single-cell expression profiling, and multiplexed quantitative single-molecule mRNA imaging in thick autofluorescent samples.\n\nSUMMARYIn situ hybridization chain reaction (HCR) v3.0 exploits automatic background suppression to enable multiplexed quantitative mRNA imaging and flow cytometry with dramatically enhanced ease-of-use and performance.

developmental biology↗

bioRxiv2017

Multidimensional quantitative analysis of mRNA expression within intact vertebrate embryos

For decades, in situ hybridization methods have been essential tools for studies of vertebrate development and disease, as they enable qualitative analyses of mRNA expression in an anatomical context. Quantitative mRNA analyses typically sacrifice the anatomy, relying on embryo microdissection, dissociation, cell sorting, and/or homogenization. Here, we eliminate the tradeoff between quantitation and anatomical context, using multiplexed in situ hybridization chain reaction (HCR) to perform accurate and precise relative quantitation of mRNA expression with subcellular resolution within whole-mount vertebrate embryos. Gene expression can be queried in two directions: read-out from anatomical space to expression space reveals co-expression relationships in selected regions of the specimen; conversely, read-in from multidimensional expression space to anatomical space reveals those anatomical locations in which selected gene co-expression relationships occur. As we demonstrate by examining gene circuits underlying somitogenesis, quantitative read-out and read-in analyses provide the strengths of flow cytometry expression analyses, but by preserving subcellular anatomical context, they enable iterative bi-directional queries that open a new era for in situ hybridization.\n\nSUMMARYMultiplexed in situ hybridization chain reaction (HCR) enables quantitative multidimensional analyses of developmental gene expression with subcellular resolution in an anatomical context.

developmental biology↗