A decade of alkyne-tag Raman imaging (ATRI): applications in biological systems

Alkyne functional groups have unique stretching frequency in the cell silent region. This review discusses the application of alkyne tags for Raman imaging in biological samples.

Quantitative analysis of Histone modifications in gene silencing

Gene silencing in budding yeast is mediated by Sir protein binding to unacetylated nucleosomes to form a chromatin structure that inhibits transcription. This transcriptional silencing is characterized by the high-fidelity transmission of the silent state. Despite its relative stability, the constituent parts of the silent state are in constant flux giving rise to a model that silent loci can tolerate such fluctuations without functional consequences. However, the level of tolerance is unknown and we developed a method to measure the threshold of histone acetylation that causes the silent chromatin state to switch to the active state. We show that loss of silencing required between 50% and 75% of the unacetylated histones to be replaced with acetylated histone mimics. The precise levels of unacetylated nucleosomes required varied from locus to locus and was influenced by both silencer strength and UAS enhancer/promoter strength. Simple calculations suggest that an approximately 50% reduction in the ability of acetylases to acetylate individual nucleosomes across a large domain may be sufficient to generate a transcriptionally silent region in the nucleus.

Polydiacetylene-based ultrastrong bioorthogonal Raman probes for targeted live-cell Raman imaging

Live-cell Raman imaging based on bioorthogonal Raman probes with distinct signals in the cellular Raman-silent region (1800–2800 cm−1) has attracted great interest in recent years. We report here a class of water-soluble and biocompatible polydiacetylenes with intrinsic ultrastrong alkyne Raman signals that locate in this region for organelle-targeting live-cell Raman imaging. Using a host-guest topochemical polymerization strategy, we have synthesized a water-soluble and functionalizable master polydiacetylene, namely poly(deca-4,6-diynedioic acid) (PDDA), which possesses significantly enhanced (up to ~104 fold) alkyne vibration compared to conventional alkyne Raman probes. In addition, PDDA can be used as a general platform for multi-functional ultrastrong Raman probes. We achieve high quality live-cell stimulated Raman scattering imaging on the basis of modified PDDA. The polydiacetylene-based Raman probes represent ultrastrong intrinsic Raman imaging agents in the Raman-silent region (without any Raman enhancer), and the flexible functionalization of this material holds great promise for its potential diverse applications.

Kinetic analysis of bioorthogonal reaction mechanisms using Raman microscopy

Kinetic analysis of CuAAC and Glaser–Hay bioorthogonal reactions can be achieved with Raman microscopy using alkyne vibrations in the cell-silent region.

LOCAL CONTROL OF SOUND IN STOCHASTIC DOMAINS BASED ON FINITE ELEMENT MODELS

A numerical method for optimizing the local control of sound in a stochastic domain is developed. A three-dimensional enclosed acoustic space, for example, a cabin with acoustic actuators in given locations is modeled using the finite element method in the frequency domain. The optimal local noise control signals minimizing the least square of the pressure field in the silent region are given by the solution of a quadratic optimization problem. The developed method computes a robust local noise control in the presence of randomly varying parameters such as variations in the acoustic space. Numerical examples consider the noise experienced by a vehicle driver with a varying posture. In a model problem, a significant noise reduction is demonstrated at lower frequencies.

The 2.1-kb Inverted Repeat DNA Sequences Flank themat2,3Silent Region in Two Species of Schizosaccharomyces and Are Involved in Epigenetic Silencing inSchizosaccharomyces pombe

The mat2,3 region of the fission yeast Schizosaccharomyces pombe exhibits a phenomenon of transcriptional silencing. This region is flanked by two identical DNA sequence elements, 2.1 kb in length, present in inverted orientation: IRL on the left and IRR on the right of the silent region. The repeats do not encode any ORF. The inverted repeat DNA region is also present in a newly identified related species, which we named S. kambucha. Interestingly, the left and right repeats share perfect identity within a species, but show ∼2% bases interspecies variation. Deletion of IRL results in variegated expression of markers inserted in the silent region, while deletion of the IRR causes their derepression. When deletions of these repeats were genetically combined with mutations in different trans-acting genes previously shown to cause a partial defect in silencing, only mutations in clr1 and clr3 showed additive defects in silencing with the deletion of IRL. The rate of mat1 switching is also affected by deletion of repeats. The IRL or IRR deletion did not cause significant derepression of the mat2 or mat3 loci. These results implicate repeats for maintaining full repression of the mat2,3 region, for efficient mat1 switching, and further support the notion that multiple pathways cooperate to silence the mat2,3 domain.