A series of dialkylformamidine protected deoxyadenosine phosphoramidites were prepared for automated, solid-support DNA synthesis. The set of Abz, Gdmf, Cbz, T phosphoramidites gave high purity and high yield oligodeoxynucleotides, with complete deprotection at 65 °C in one hour. Different times and temperatures of exposure to concentrated ammonium hydroxide were examined to establish the optimum conditions for deprotection of oligodeoxynucleotides.

N-6-Dialkylformamidine-2′-deoxyadenosine Phosphoramidites in Oligodeoxynucleotide Synthesis Raped Deprotection of Oligodeoxynucleotides

Oxidative conversion of N-dimethylformamidine nucleosides to N-cyano nucleosides

Synthetic nucleic acids are produced routinely for a wide variety of applications, including biological and chemical research, and diagnostic or therapeutic applications. To ensure an adequate level of quality and purity, rapid and convenient analytical methods are necessary. This unit discusses basic principles to guide in the selection of appropriate purification and analysis protocols.

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Overview of purification and analysis of synthetic nucleic acids.

1 Chemical Synthesis of Oligonucleotides: From Dream to Automation

The mechanical properties of biological tissues are key to their physical integrity and function. Although external loading or biochemical treatments allow the estimation of these properties globally, it remains difficult to assess how such external stimuli compare with cell-generated contractions. Here we engineer microtissues composed of optogenetically-modified fibroblasts encapsulated within collagen. Using light to control the activity of RhoA, a major regulator of cellular contractility, we induce local contractions within microtissues, while monitoring microtissue stress and strain. We investigate the regulation of these local contractions and their spatio-temporal distribution. We demonstrate the potential of our technique for quantifying tissue elasticity and strain propagation, before examining the possibility of using light to create and map local anisotropies in mechanically heterogeneous microtissues. Altogether, our results open an avenue to guide the formation of tissues while non-destructively charting their rheology in real time, using their own constituting cells as internal actuators. 

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Light-driven biological actuators to probe the rheology of 3D microtissues

AutoAnalysis is a new method for detecting and quantitating the trityl cation released each cycle on automated DNA synthesizers. The trityl (dimethoxytrityl) cation is removed from the growing oligonucleotide after each base addition and is a useful measure of synthesis efficiency. The traditional absorbance method of collecting each trityl effluent with a fraction collector, followed by dilution with an acid solution and careful quantitation by UV/VIS spectroscopy is costly, tedious and prone to error. The absorbance method for trityl cation analysis must usually wait until the synthesis is complete. Interruption of a failed operation, for a variety of reasons, such as an empty reagent reservoir, is thus not possible. Taking advantage of the conductive properties of the trityl cation, immediate and real-time quantitation is now possible by integrating the total conductance of the flowing stream during the detritylation step after each nucleoside addition in DNA synthesis. A conductivity cell is mounted downstream, past the synthesis column. The conductivity signal is processed and displayed as the current average stepwise yield and overall yield. If the yield drops below a pre-set threshold value because of a failure situation, the synthesizer will interrupt, preserving reagents. AutoAnalysis allows trityl monitoring with complete automation on the Applied Biosystems Models 392 and 394 DNA/RNA Synthesizers.

Trityl monitoring of automated DNA synthesizer operation by conductivity : a new method of real-time analysis

Cell size and actin architecture determine force generation in optogenetically activated cells.

Adherent cells use actomyosin contractility to generate mechanical force and to sense the physical properties of their environment, with dramatic consequences for migration, division, differentiation and fate. However, the organization of the actomyosin system within cells is highly variable, with its assembly and function being controlled by small GTPases from the Rho-family. How activation of these regulators translates into cell-scale force generation and the corresponding sensing capabilities in the context of different physical environments is not understood. Here we probe this relationship combining recent advances in non-neuronal optogenetics with micropatterning and traction force microscopy on soft elastic substrates. We find that after whole-cell RhoA-activation by the CRY2/CIBN optogenetic system with a short pulse of 100 milliseconds, single cells contract before returning to their original tension setpoint with near perfect precision on a time scale of several minutes. To decouple the biochemical and mechanical elements of this response, we introduce a mathematical model that is parametrized by fits to the dynamics of the substrate deformation energy. We find that the RhoA-response builds up quickly on a time scale of 20 seconds, but decays slowly on a time scale of 50 seconds. The larger the cells and the more polarized their actin cytoskeleton, the more substrate deformation energy is generated. RhoA-activation starts to saturate if optogenetic pulse length exceeds 50 milliseconds, revealing the intrinsic limits of biochemical activation. Together our results suggest that adherent cells establish tensional homeostasis by the RhoA-system, but that the setpoint and the dynamics around it are strongly determined by cell size and the architecture of the actin cytoskeleton, which both are controlled by the extracellular environment.

Fitzpatrick

Papers

Trityl monitoring of automated DNA synthesizer operation by conductivity : a new method of real-time analysis

Cell size and actin architecture determine force generation in optogenetically activated cells.

Cell size and actin architecture determine force generation in optogenetically activated adherent cells