Transcriptome in vivo analysis (TIVA) of spatially defined single cells in live tissue.

Transcriptome profiling of single cells resident in their natural microenvironment depends upon RNA capture methods that are both noninvasive and spatially precise. We engineered a transcriptome in vivo analysis (TIVA) tag, which upon photoactivation enables mRNA capture from single cells in live tissue. Using the TIVA tag in combination with RNA sequencing (RNA-seq), we analyzed transcriptome variance among single neurons in culture and in mouse and human tissue in vivo. Our data showed that the tissue microenvironment shapes the transcriptomic landscape of individual cells. The TIVA methodology is, to our knowledge, the first noninvasive approach for capturing mRNA from live single cells in their natural microenvironment.

Caged oligonucleotides for bidirectional photomodulation of let-7 miRNA in zebrafish embryos.

Many biological functions of microRNA (miRNA) have been identified in the past decade. However, a single miRNA can regulate multiple gene targets, thus it has been a challenge to elucidate the specific functions of each miRNA in different locations and times. New chemical tools make it possible to modulate miRNA activity with higher spatiotemporal resolution. Here, we describe light-activated (caged) constructs for switching let-7 miRNA 'on' or 'off' with 365 nm light in developing zebrafish embryos.

Mismatch Discrimination and Efficient Photomodulation with Split 10-23 DNAzymes.

DNA enzymes (DNAzymes) that catalyze the degradation of complementary RNA molecules have been investigated for many biochemical and sensing applications. Here, we investigated a 10-23 DNAzyme that has been shown previously to possess cellular activity. We determined that it has very low Mg(2+) ion dependence, with DNAzyme activity observed at [Mg(2+)] = 0.01 mM. This metal ion dependence is much lower than is typical for DNAzymes studied to date, and suggests that DNAzymes may be engineered for many additional biological applications. Recently, we demonstrated that this 10-23 DNAzyme can be divided into two parts, which assemble into an active oligonucleotide complex. We investigated in more detail the functionality of the split 10-23 DNAzyme and found that dividing the 15-nucleotide catalytic loop after the 7(th) or 8(th) base maximized its activity. The split DNAzymes required higher metal ion concentrations ([Mg(2+)] = 5 mM), and as we anticipated due to their lower hybridization activity, the split enzymes had the advantage of being more sensitive to single base mismatches in the DNAzyme-RNA duplex. Finally, we demonstrated facile photomodulation of split DNAzyme activity by incorporating a photocleavable biotin moiety bound to streptavidin.

Caged oligonucleotides for studying biological systems.

Light-activated ("caged") compounds have been widely employed for studying biological processes with high spatial and temporal control. In the past decade, several new approaches for caging the structure and function of DNA and RNA oligonucleotides have been developed. This review focuses on caged oligonucleotides that incorporate site-specifically one or two photocleavable linkers, whose photolysis yields oligonucleotides with dramatic structural and functional changes. This technique has been employed by our laboratory and others to photoregulate gene expression in cells and living organisms, typically using near UV-activated organic chromophores. To improve capabilities for in vivo studies, we harnessed the rich inorganic photochemistry of ruthenium bipyridyl complexes to synthesize Ru-caged morpholino antisense oligonucleotides that remain inactive in zebrafish embryos until uncaged with visible light. Expanding into new caged oligonucleotide applications, our lab has developed Transcriptome In Vivo Analysis (TIVA) technology, which provides the first noninvasive, unbiased method for isolating mRNA from single neurons in brain tissues. TIVA-isolated mRNA can be amplified and then analyzed using next-generation sequencing (RNA-seq).