key: cord-0286940-ubixo05j
authors: Laos, Roberto; Benner, Steven
title: Fluorinated oil-surfactant mixtures with the density of water: artificial cells for synthetic biology
date: 2021-05-17
journal: bioRxiv
DOI: 10.1101/2021.05.17.444437
sha: 0c030651ceb937b09d37016ca2bcc65f28b96bde
doc_id: 286940
cord_uid: ubixo05j

Water-in-oil emulsions provide matrices for compartments that have many uses in diversity science. However, hydrophobic species are frequently incompatible with biological systems. For this reason, fluorinated matrices are often sought, since fluorinated species are neither hydrophilic nor hydrophobic; they therefore do not interact with most biomolecules. However, most fluorinated oils have densities much higher than the density of water (1 g/ml). Consequently, water droplets float in fluorinated oils, aggregating near their surfaces. This facilitates droplet-droplet collision and fusion, exposing droplets to air interfaces and making their manipulation difficult. Here, we report the synthesis, characterization, and use of fluorinated polysiloxane oils that have densities close to the density of water. These, with a non-ionic fluorosilicone surfactant, produce thermostable water-in-oil emulsions that neither float nor sink. We show how droplets in these emulsions can host many biological processes, including PCR, DNA origami, rolling circle amplification (RCA), and Taqman® assays. Further, oil-diffusible reagents can initiate reactions within the droplets. The droplets can also be used with unnatural DNA emerging from synthetic biology, including DNA built from artificially expanded genetic information systems (AEGIS) with six nucleotide “letters”.

The densities of the various fractions were measured using a balance and positive 134 displacement pipette. A glass vial and a pipette tip were placed in a balance and 135 zeroed. Then, fluid (100 µL) was taken into the pipette and the tip was cleaned with a 136 wipe. The liquid and the pipette tip were then placed on the glass vial and weighed 137 again. This was done three times; the average is reported.

138 Protocol 2. The fluorinated monomer 1,3,5trimethyl 1,3,5-tris(3,3,3-trifluoropropyl)

139 cyclotrisiloxane (25.26 g; 53.9 mmol) was mixed with the non-fluorinated monomer, (Fig 3) . This allowed the RCA to continue.

274 Evidence for successful RCA came by measuring fluorescence for 10 seconds after 

The lysate was then loaded on a column and washed with: (a) 2 mL wash: Tris, 20 293 mM; NaCl, 1 M; followed by (b) 1 mL wash: Tris, 20 mM; NaCl, 150 mM; followed by (c) 

With these considerations, we explored methods to prepare low-density fluorous oils 309 by reacting siloxanes having both alkane and trifluoromethylalkane side chains, 310 specifically 1,3,5-trimethyl 1,3,5-tris(3,3,3-trifluoropropyl)-cyclotrisiloxane and 311 hexamethylcyclotrisiloxane (Fig 1) . (Table 3) . We found that the nonionic fluorinated surfactant shown is soluble 392 up to 5% in the low molecular weight fraction while it is not soluble in the high molecular 393 weight fraction. We found useful to combine four volumes of the high molecular weight 394 fraction with 1 volume of the low molecular weight fraction obtained using protocol 1.

395 Using one particular batch 4 mL of a HMW fraction (density: 0.984 g/mL) was combined 396 with 1 mL of the LMW fraction (density: 1.0613 g/mL) to produce a fluid of density 1.009 397 g/mol where we could solubilize 2% of the fluorinated surfactant without visible 398 separation of the surfactant from the fluid.

The preferred oils were then challenged to support processes that involve both DNA 400 and enzymes, all useful in biomolecular sciences, nanotechnology, and diversity 

filters into autosampler vials, filled to the shoulder

The origami folding mixture comprised 208 staples, each 200 nM (Table in S1 Table) 197 and a scaffold (20 nM)

Tris buffer (5 mM, pH ~ 8.2, with 1 mM EDTA, 20 mM 199 MgCl 2 , and 5 mM NaCl

Droplet formation was done using PDMS radial chips that empty droplets of aqueous

1) with the fluorinated surfactant 2% (v/v)

The origami mixture, both bulk (as a positive control) and emulsified, were placed in a 206 thermal cycler at 65° C for 10 minutes and then cooled to 60° C at 0.1° C/s. After this 207 hold, the temperature was lowered by 0.5° C steps (0.1° C/s rate) and held at the new 208 temperature for 30 minutes until 40° C was reached

An aqueous solution holding all components required for a PCR was prepared, 217 consisting of: (a) primers (20 nM each) 5'-GACGGACTGCCTATGAG-3 and 5'-218 GAGGCGATCGCAGTATC-3'; (b) double stranded template

2 mM each), (d) Evagreen 2X; (e) a variant 222 DNA polymerase

µM; and (f) buffer, with 20 mM Tris-HCl

The mixture was dispersed into the fluorous oil emulsion at 25°C, again using a

2 µM concentration.; and 237 • Probe: 5'-FAM-ATGAATACC-ZEN-TTTGTTCTCTGCTGG CAACTGCTG-3' 238 The other components of the reaction mixture were: (a) substrate dNTPs and dZTP, 239 each at 0.2 mM; (b) buffer at 10 mM Tris pH 8.3, 1.5 mM MgCl 2 , 50 mM KCl; and (c) 240 enzyme, a variant of Taq DNA polymerase

The aqueous mixture (100 μL) was emulsified into fluorinated oil (300 μL) with 2%

An aqueous mixture comprising all of the components for a RCA reaction was 252 prepared to include: (a) 1X Phi29 buffer

NH 4 ) 2 SO 4 , and 4 mM DTT (@ 25°C); (b) 254 triphosphates, dATP, dCTP, dTTP 0.2 mM each; (c) a 3'-blocked triphosphate

The aqueous mixture holding all the components necessary for RCA

Generation of fluorescent signals inside droplets suspended in the fluorinated oil 409 using blocked nucleoside triphosphate's, where the reaction is initiated by adding a 410 reagent (amyl nitrite) that is able to diffuse through the fluorinated, enter the droplet, and and 6 mM MgCl 2 . The "nanostructure PF-2, cuboid with large aperture

To demonstrate that droplets suspended in the iso-dense fluorinated oil had thermal 436 stability adequate to support PCR and

with the N-terminus of the catalytic domain fused to a processivity enhancing 441 domain from Sulfolobus solfataricus. The Taq variant was selected using diversity 442 science, and has 6 mutations, two specific for enhancing the incorporation of the pair 443 Z:P. The enzyme is described as

Progress of the PCR was monitored by observing the fluorescence of Evagreen ® , 446 which intercalates into double-stranded PCR amplicons. Fig 7 shows real time PCR 447 fluorescence signals arising from a PCR done, as a control

PCR done in parallel in water droplets emulsified in iso-dense fluorinated oil. The Ct

Evagreen ® without emulsification (blue line) and in the emulsified PCR (red line). The 463 emulsion was prepared using microfluidic devices made of PDMS. The aqueous phase 464 was delivered (25°C) at 5 µL/min by the PDMS chip into the fluorinated oil matrix at 12

The matrix phase is a fluorinated silicon polymer with 2% of surfactant (see Figs 466 1 and 2 for structures

One standard way to detect DNA is a Taqman ® assay. Here, DNA polymerase from

Thermus aquaticus degrades the downstream strand using its 5'-3' exonuclease activity 474 displacing a probe from the template, cleaving the probe in the process. This cleavage 475 separates a fluorescent reporter (here, fluorescein, FAM) from one end of the probe 476 from a fluorescence quencher at the other end of the probe

Here, we adapted the Taqman ® assay to detect a target analyte that contained AEGIS

Fig 9 shows in real-time fluorescence detected in that assay. Again, output was

Bottom: Real-time fluorescence detected 492 from FAM probe when Taq [R587Q E832C] polymerase places a dZ opposite dP. The 493 blue line is the fluorescence from a non-emulsified reaction. The red line corresponds to 494 the signal from the emulsified reaction. The emulsion was made by mixing 100 μL of 495 aqueous phase with 300 μL of a

Many regard water droplets in compartments as inaccessible to reagent-based 502 manipulation. Here, we overcame this limitation by showing that reactions can be 503 initiated within droplets isolated in fluorinated oil by adding "turn on" reagents that can 504 diffuse through the oil. This allowed us to mix DNA templates

With a 3'-O-NH 2 blocking group, primers cannot be extended 509 by a DNA polymerase. However, treatment with amyl nitrite cleaves the O-N bond to 510 yield an extendable 3'-OH group. Further, we discovered that amyl nitrite will diffuse 511 through the fluorinated oil and partition

RCA works by having a circular template, a primer that binds to that circular 515 template, and a strand displacing polymerase that extends that primer, displacing 516 already synthesized DNA as it proceeds around the circle. Here, the aqueous phase 517 used to prepare the droplets contains a circularized template 96 nucleotides around, a 518 primer complementary to the circular template, and three natural dNTPs (dATP, dCTP 519 and dTTP)

200 µL of a high molecular 522 weight fraction of the fluorinated oil containing amyl nitrite (0.1% v/v). The progress of in droplets. Fluorescence detection is acquired after each two step cycle 529 [37 C for 60 seconds-37 C for10 seconds followed by aquisition] x 99 cycles

Emulsion using all natural dNTPs. Initiator added to 0.08%. Line C. Emulsion 532 containing dATP, dCTP, dTTP and dGTP-ONH 2

Line D: Emulsion containing dATP, dCTP, dTTP and dGTP-ONH 2 . No amyl nitrite

The growing interest in synthetic cells [49] matches a growing interest in engineering 538 enzymes for synthetic biology [50, 51] [52] by directed evolution. For example, Tawfik 539 and Griffiths [24] developed a method to link genotype and phenotype in droplets of 540 water emulsified in oil called

One of the first problems identified was associated to the surfactant Span 545 80 ® [53

Thus, while results are possible with surfactant mixtures containing Span 80 ® , 549 these are hit-or-miss, and this corner of biotechnology has become known in the 550 community as problematically irreproducible. Further, although they are not frequently 551 published, many observations suggest that some biological systems suffer denaturation 552 when they encounter hydrocarbon oils

For this reason, many have turned to the synthetic materials and a third liquid phase, 554 the "fluorous phase

Here, balancing the mixture of hydrocarbon-carrying silanes, intrinsically less dense 562 than water, with fluorohydrocarbon-carrying silanes

Multiple standard challenges in nanotechnology were applied to these iso-dense 567 fluorinated oil-surfactant systems: (a) origami, (b) PCR, (c) Taqman® assays, and (d)

All of these biochemical reactions worked as well in these iso-dense fluorinated 569 oil-surfactant systems as their non-emulsified analogs. Thus

Sobczak from Tilibit Nanosystems for 573 useful discussions and assistance in troubleshooting and confirming the formation of the 574 origami structure. We thank Brian Paegel laboratory for providing molds and assistance 575 in preparing microfluidic devices as well as valuable discussions in surfactant design

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