Nanodroplets persisted in solution longer than microbubbles.

<p><b>A)</b> Flow chart outlining method for production of nanodroplets. <b>B)</b> A persistence study was performed in the ultrasonic bath to compare nanodroplets and microbubbles. An Accusizer particle sizing system (Particle Sizing Systems, Port Richey, FL) was used to measure the microbubble and nanodroplet concentrations at specific time points between 0 and 300 seconds (5 minutes). Nanodroplets maintained between 10–20% of their initial concentration as far out as 3 minutes into the sonication treatment, while the microbubble concentration dropped to 10% after 1 second.</p

ATP K_m determination for TBK1 and IKKε.

<p>Enzymatic reactions of A) TBK1 or B) IKKε were incubated at room temperature with 10 ATP concentrations varying from 333 µM to 0.017 µM in three fold dilutions. Reactions were sampled on the Caliper EZReader system at 9.35 minute intervals over a 3 hour period. Percent conversions were calculated from relative heights of product and substrate peaks and used to calculate velocity and ATP K_m in Graph Pad Prism.</p

Cavitation Enhancing Nanodroplets Mediate Efficient DNA Fragmentation in a Bench Top Ultrasonic Water Bath

<div><p>A perfluorocarbon nanodroplet formulation is shown to be an effective cavitation enhancement agent, enabling rapid and consistent fragmentation of genomic DNA in a standard ultrasonic water bath. This nanodroplet-enhanced method produces genomic DNA libraries and next-generation sequencing results indistinguishable from DNA samples fragmented in dedicated commercial acoustic sonication equipment, and with higher throughput. This technique thus enables widespread access to fast bench-top genomic DNA fragmentation.</p></div

Identification of the optimal phosphorylation motif for TBK1.

<p>A-B) The positional scanning peptide library technology was used to determine the optimal phosphorylation motif for recombinant A) GST-TBK1 WT or B) kinase-dead GST-TBK1 K38A as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041494#pone.0041494-Hutti3" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041494#pone.0041494-Turk1" target="_blank">[28]</a>. Briefly, 198 peptide libraries were phosphorylated in individual kinase assays. The sequence for these libraries is Y-A-X-X-X-Z-X-S/T-X-X-X-X-A-G-K-K-biotin (Z =  fixed amino acid, X =  equimolar mixture of amino acids excluding Ser, Thr, and Cys). After binding to a streptavidin-coated membrane, phosphorylation was visualized by the incorporation of ³²P. C) Primary and secondary selections for TBK1, as determined in A). D) 50 µM of the indicated peptide was phosphorylated in an <i>in vitro</i> kinase assay with recombinant GST-TBK1 for 30 min. Phosphorylation of each peptide is shown as a percentage of the rate of phosphorylation of TBK1-Tide, the optimal peptide substrate for TBK1. Error bars are standard deviation.</p

<i>Saccharomyces cerevisiae</i> gDNA (BY4741) fragmented with nanodroplets in an ultrasonic bath was comparable in quality to DNA fragmented using a commercial method.

<p><b>(A)</b> Agilent D1000 ScreenTape system traces for DNA samples in microTUBES with the rod (left panel) or microTUBES with nanodroplets (right panel) that were subjected to sequencing. Average size is indicated in base pairs (bp). DNA size markers are denoted by Upper and Lower. <b>(B)</b> Traces showing similar size distribution of DNA after sequencing library preparation. Average size is indicated in base pairs (bp). DNA size markers are denoted by Upper and Lower. <b>(C)</b> Mapping sequencing reads to the <i>Saccharomyces cerevisiae</i> (S288c) reference genome is comparable in detection of single nucleotide variations, insertions, and deletions. Abundance and profile of relative errors in sequencing reads does not indicate a difference in the presence of error bias in the data.</p

Nanodroplets were an effective cavitation agent for use in DNA fragmentation.

<p><b>A)</b> The effectiveness of nanodroplets as a cavitation enhancement agent after multiple freeze-thaw cycles was tested. DNA ladder size is indicated in base pairs. Input is DNA prior to sonication with nanodroplets. <b>B)</b> Comparison of DNA fragmentation efficiency after two minutes in glass (Lanes 1–3) versus plastic (Lanes 4–6) tubes in the Covaris E110 sonicator. The addition of nanodroplets to Covaris microTUBES produces a DNA fragment size distribution comparable to the microTUBES used with the supplied rod (compare Lanes 1 and 3). DNA fragmented in glass microTUBES had a smaller DNA size distribution compared to plastic 0.2 mL PCR tubes (compare Lanes 3 and 5–6). DNA ladder size is indicated in base pairs.</p

Nanodroplet-mediated DNA fragmentation compared to a commercial method.

<p><b>(A)</b> Flow chart outlining method for comparing DNA fragmentation methods. <b>(B)</b> False gel picture from Agilent D1000 ScreenTape system showing DNA fragment size distribution in base pairs for samples fragmented in the Covaris E110 sonicator. Purple bars indicate the upper (1,500 bp) molecular weight marker and green bars indicate the lower (25 bp) molecular weight marker in each lane.</p

The use of nanodroplets allowed an ultrasonic water bath to fragment genomic DNA.

<p><b>(A)</b> Schematic showing the ultrasonic bath used for sonication. Samples were immobilized in the water bath using a stand with a tube rack attached. The circulating water chiller was optional. Water chilled to four degrees Centigrade can be added just prior to sonication, with no loss in DNA fragmentation efficiency. <b>(B)</b> A time-titration was performed with samples with and without nanodroplets. Following fragmentation, samples were run on a 1.5% agarose gel and visualized using SYBR green. DNA ladder sizes are indicated in base pairs. <b>(C)</b> Arrangement of DNA samples fragmented in the ultrasonic bath with and without samples to produce <b>(D)</b> an acoustic field map of the bath. The fragmentation ability (base pair size) is visualized with the s1color bar, where red indicates complete fragmentation in the 200–500 bp range.</p

DNA fragmentation in an ultrasonic water bath compared to a commercially available device.

<p><b>(A)</b> Flow chart outlining method for comparing DNA fragmentation methods.</p

Activity comparison for TBK1 and IKKε.

<p>Number of active compounds (N) detected in each screen for total number detected (unfiltered), number after drug like filtering (filtered.drug-like), hits from the LOPAC and Kinase libraries, and the number of chemical clusters and singleton hits as described in the text.</p