Fig 1.
The Z:P pair from an Artificially Expanded Genetic Information System (AEGIS), left.
AEGIS nucleotides follow the geometry and hydrogen bonding architecture of the natural nucleotides. The hydrogen bonding patterns are named pu or py, depending on whether they are presented on a large (purine-like) or small (pyrimidine-like) heterocyclic ring system respectively. Hydrogen bond donor (D) and acceptor (A) groups listed starting in the major groove and ending in the minor groove. Unshared pairs of electrons presented to the minor groove are shown in green orbitals. The Z:P pair presents the pyDDA:puAAD hydrogen bonding pattern, while the analog C:G pair presents the pyDAA:puADD. R is deoxyribose.
Fig 2.
Ring-opening polymerization used to produce the fluorosilicone fluid.
The two cyclotrisiloxane monomers (hexamethylcyclotrisiloxane and 1,3,5-trimenthyl 1,3,5-tris(3,3,3-trifluoropropyl)cyclotrisiloxane) are reacted along with hexamethyl disiloxane, which acts as a chain terminator. The catalyst Amberlyst® 15 acts as an acid catalyst for the ring-opening reaction.
Fig 3.
Nonionic fluorosilicone surfactants.
The amphiphilic molecules are produced by reacting SiH functionalized branched disiloxanes hydrophobes with allyloxy(polyethylene oxide) following reference [50]. Two different hydrophiles (a, b) and two hydrophobes are shown: one with the 3,3,3-trifluoropropyl (R1, Prf) and 3,3,4,4,5,5,6,6,6-nonafluorohexyl (R2, Hxf).
Fig 4.
Molecular weight distribution of polysiloxanes produced via protocol 1.
Ring-opening polymerization following “protocol 1” produced polysilioxanes with bimodal distribution. These two peaks can be separated by distillation under reduced pressure with a high molecular weight peak (Mp ~ 9,400 g/mol) and a low molecular weight peak (Mp ~ 300 g/mol). The low molecular weight fraction contains a higher concentration of fluorinated monomers estimated by 1H-NMR and is capable of dissolving the fluorinated surfactants. Peak 1 corresponds to the higher molecular weight peak (rightmost) and peak 2 corresponds to the lower molecular weight peak (leftmost). S4 Fig in S3 File. shows an overlay of two gel permeation chromatography runs of the two distinctive fractions separated by distillation.
Fig 5.
Molecular weight distribution of polysiloxanes produced via protocol 2.
Ring-opening polymerization following “protocol 2” produced two liquid polysilioxanes that spontaneously separate in two phases with different densities and are referred as fluid C (red) and fluid D (blue). Each fluid exhibits bimodal molecular weight distribution.
Table 1.
Summary of molecular weight moments and polydispersity.
Fig 6.
1NMR analysis of fluorosilicone polysiloxanes.
Overlay of the 1HNMR of the low molecular weight (blue) and the high molecular weight (red) fraction samples indicating the assigned peaks.
Table 2.
Integration of peaks used for determination of the ratio of non-fluorinated monomer: Fluorinated monomer.
Fig 7.
19FNMR overlays showing the presence of the surfactant with the Hxf hydrophobe CF3CF2CF2CF2CH2CH2- dissolved in the low molecular weight fluid (fluid B).
(A) A single peak that comes from the CF3CH2CH2- from the fluorinated fluid. (B) Four peaks that correspond to the fluorine of the Hxf hydrophobe CF3CF2CF2CF2CH2CH2-. (C) 19FNMR of a homogeneous 4% solution (v/v) of the surfactant in the fluorinated fluid exhibiting the signals from both components.
Fig 8.
1HNMR overlays showing the presence of the surfactant with the Prf hydrophobe CF3CH2CH2- dissolved in the low molecular weight fluid (fluid B).
(A) 1HNMR of the fluid without surfactant. (B) 1HNMR of a 4% (v/v the fluid with surfactant.A single peak that comes from the CF3CH2CH2- from the fluorinated fluid. The characteristic signals of the -(CH2CH2-O)- can be observed at 3.4–3.8 ppm.
Table 3.
Viscosity, density and other properties measured on fluorinated polysiloxanes.
Fig 9.
Transmission microscope images of droplets containing oligos with a FAM fluorescent molecule or a Black Hole Quencher (BHQ).
The fluorinated fluid (fluid B) with 1% of surfactant (b-R1) was pumped at 10 μL/min. Aqueous phase contained 5 μM of 5’-CTGCTCATAGG CAGTCCGTCA-BHQ-3’ or 5’-FAM-TGACGGACTGCCTATGAGCAG-3’ in 20 mM Tris-HCl, pH 8.4; 10 mM (NH4)2SO4; 10 mM KCl; 2 mM MgSO4; 0.1% Triton® X-100. Aqueous phase was pumped at 2 μL/min. (A) Droplets carrying oligo with Black Hole Quencher (BHQ). (B) Droplets carrying oligo with FAM. (C). Droplets carrying BHQ placed on the left side of the glass slide and droplets carrying FAM placed on the right hand side of the slide. (D) After being in contact for 10 minutes no fusion is observed. Scale bar is 400 μm. Pictures are representative of several trials.
Fig 10.
Emulsion made with fluid C (protocol 2).
Left: transmission microscope image of droplets. Scale bar is 200 μm. Right: picture of the emulsion formed by shaking 100 μL of buffer and 300 μL of fluid C with 2% of the surfactant a-R2 containing the Hxf hydrophobe. The emulsion is formed by shaking the contents in a Snap-Cap Microcentrifuge Biopur™ Safe-Lock™ Tube containing a 6 mm stainless steel bead. The tube was fitted in a TissueLyser II and shaken for 10 seconds at 15 Hz followed by 7 seconds at 17 Hz. Scale bar is 400 μm. Pictures are representative of several trials.
Fig 11.
DNA electrophoresis in agarose (2%) showing the formation of origami structures in droplets.
The origami products were run in 0.5 X Tris-borate EDTA buffer with ethidium bromide and 6 mM MgCl2. The origami structure folded is called “nanostructure PF-2, cuboid with large aperture” from Tilibit Nanosystems. The scaffold, a single-stranded DNA type p7249 isolated from M13mp18 of length 7,249 bases. The staples are a mixture of 208 oligos (S1 Table in S3 File). A sample of the mixture of scaffold and staples not annealed is run for comparison. The origami folded properly shows a distinctive migration on the gel (see S8 and S9 Figs for reference in S3 File). Annealing was done by incubating at 65°C for 10 minutes then cooled down from 60°C to 40°C at a rate of 1°C per hour.
Fig 12.
Real time PCR showing emergence of fluorescence from intercalated Evagreen® without emulsification (blue lines) and in the emulsified PCR (red lines).
The aqueous phase was emulsified by mixing 100 μL of aqueous phase mixed with 300 μL of fluorinated oil (1 volume of LMW fraction and 4 volumes of the HMW fraction, see protocol 1) with 2% of surfactant (a-R2). Assays done by triplicate.
Fig 13.
Top: Scheme of the Taqman® assay containing AEGIS (dZ:dP pair hydrogen bonding shown). Bottom: Real-time fluorescence detected from a probe. The probe contains a 5′ fluorophore: 6-carboxyfluorescein (6FAM) and is double-quenched with: 3′ Iowa Black™ (IABk) and a proprietary internal ZEN quencher (IDT). When Taq [R587Q E832C] polymerase places a dZ opposite dP and the 5’-3’ exonuclease of Taq polymerase cleaves the probe eventually releasing a fluorescent molecule. The blue line is the fluorescence from a non-emulsified reaction. The red line corresponds to the signal from the emulsified reaction. The emulsion was made by mixing 100 μL of aqueous phase with 300 μL of a fluorinated oil with 2% (v/v) fluorinated surfactant as shown on Fig 2. Mixed in QIAGEN TissueLyser II in the presence of a 6 mm stainless steel bead at 15 Hz for 10 seconds followed by 17 Hz for 7 seconds using an Eppendorf Safe-Lock Tube (2 mL) containing a stainless steel ball. Assays were done by triplicate.
Fig 14.
Amplification curves obtained using the LightCycler96 for RCA amplification in droplets.
Fluorescence detection (EvaGreen®) is acquired after each two step cycle [37°C for 30 seconds—37°C for 30 seconds followed by aquisition] x 60 cycles. Blue lines correspond to the reaction that was not emulsified. Red lines correspond to the emulsified reaction. Assays done by triplicate.