Spatiotemporal Regulation of Cell Fate in Living Systems Using Photoactivatable Artificial DNA Membraneless Organelles

Coacervates formed by liquid–liquid phase separation emerge as important biomimetic models for studying the dynamic behaviors of membraneless organelles and synchronously motivating the creation of smart architectures with the regulation of cell fate. Despite continuous progress, it remains challenging to balance the trade-offs among structural stability, versatility, and molecular communication for regulation of cell fate and systemic investigation in a complex physiological system. Herein, we present a self-stabilizing and fastener-bound gain-of-function methodology to create a new type of synthetic DNA membraneless organelle (MO) with high stability and controlled bioactivity on the basis of DNA coacervates. Specifically, long single-strand DNA generated by rolling circle amplification (RCA) is selected as the scaffold that assembles into membraneless coacervates via phase separation. Intriguingly, the as-formed DNA MO can recruit RCA byproducts and other components to achieve self-stabilization, nanoscale condensation, and function encoding. As a proof of concept, photoactivatable DNA MO is constructed and successfully employed for time-dependent accumulation and spatiotemporal management of cancer in a mouse model. This study offers new, important insights into synthetic membraneless organelles for the basic understanding and manipulation of important life processes.

Fluorescence characterizations were carried out on a FV1000 confocal laser scanning microscope.A JEM-2100 Plus transmission electron microscope and a SUPRA 40 scanning electron microscope were employed for morphology characterization of the products.Element analysis of the samples was performed with inductively coupled plasma optical emission spectroscopy (ICP-OES) and transmission electron microscopy energy-dispersive X-ray spectroscopy (TEM-EDS).

LLPS-driven condensation of metastable long ssDNA membraneless organelles (mLDMO)
The mLDMO was constructed according to a reported reference. 1The phosphorylated linear template DNA and primer DNA were mixed at 1 μM in 100 μL of 1 x DNA ligation buffer (5 mM Tris-HCl, 1 mM MgCl2, 0.1 mM ATP, and 1 mM DTT).The mixed solution was then heated to 95 °C for 5 min and cooled down to 25 °C at 1 °C/s.
After annealing, 10 μL of E. coli DNA Ligase (60 U/μL) were added into the tube, gently mixed, and reacted for 3 h at 16 °C.Thereafter, the enzyme was deactivated through heating the mixture for 20 min at 65 °C.
Next, 20 μL of Exonuclease I (20 U/μL) and 20 μL of Exonuclease III (100 U/μL) were added to the mixture, and the reaction was allowed to proceed overnight at 37 °C, followed by removing the unreacted strands and linear templates.The enzymes were then deactivated by heating at 80 °C for 30 min.The template DNA was purified by an ultrafiltration device (10 kDa, Merck Millipore) and washed three times with 400 μL of TE buffer.The as-obtained ssDNA was quantified by a Nanodrop 2000 (Fisher Scientific) and diluted to 1 μM with TE buffer.
The reaction was kept at 30 °C for 60 h before heat-induced deactivation for 10 min at 70 °C.Then, the RCA products were purified by filtration with 30 kDa Millipore three times using 400 μL of TE buffer.The concentration of the long ssDNA was determined by using a Nanodrop 2000.
The mLDMO was prepared by mixing long ssDNA at the concentration of 0.2 g/L in TE buffer with 50 mM MgAc2, followed by heating to 95 °C for 15 min and cooling down to 25 °C at 1 °C/s.
The scattering of long ssDNA, mLDMO and sLDMO was first detected by a UV2450 ultraviolet spectrophotometer (Shimadzu Co., Kyoto, Japan).

Fastener-bound condensation of DNA MO
To endow DNA MO with spatiotemporally controlled activity, NIR absorbing Pd NPs were prepared according to a previous method and used as the photoresponsive fastener. 3Briefly, 50 µL of Pd NPs (0.5 mg/mL) were first washed with 500 µL acetone.
After centrifugation at 7000 rpm for 2 min, Pd NPs were redispersed in 10 µL fresh deionized water.5 µL of Pd NPs aqueous solution were mixed with 5 µL of mLDMO or sLDMO for 48 h to form photoactivatable coacervates (pmLDMO or psLDMO).
To investigate the stability of pmLDMO and psLDMO, they were labeled with Cy5 under the same procedures as noted above.The resulting pmLDMO/Cy5 or psLDMO/Cy5 was dispersed in 100 µL of PBS and imaged at the indicated incubation time (0, 0.5 h, 2 h, 4 h, 12 h, and 24 h) by CLSM.

Agarose gel electrophoresis
The stepwise formation of long ssDNA, mLDMO, and sLDMO was evaluated by 2% agarose gel electrophoresis for 30 min (120 V).After staining with Gel Red, the gel was imaged with a Gel Doc XR system (Bio-Rad).

NIR photoresponsivity of DNA MO
To study the NIR photoresponsive activity of DNA MO, FAM and Cy5 were loaded in psLDMO, and the NIR photoresponsivity of these MO was studied by fluorescence imaging.Briefly, 4 µL of FAM-DNA (100 µM) were mixed with 20 µL of psLDMO, followed by incubation with 20 µL of Pd NPs aqueous solution for 48 h.After washing with PBS three times, 4 µL of the Cy5-NH2 solution (1 mg/mL) and the resulting psLDMO/FAM were mixed in 100 µL of PBS for 24 h at 37 o C to yield psLDMO/FAM/Cy5.Thereafter, 200 µL of the resulting psLDMO/FAM/Cy5 (0.02 µM) were exposed to an 808 nm laser (100% of the maximum intensity ≤ 10 W/cm 2 ) for 1 s. 1 After centrifugation, fluorescence of the supernatant was measured to determine the release of Cy5 from psLDMO/FAM/Cy5, while the pellet was imaged by CLSM.This process was repeated five times.As a negative control, Cy5 release from Pd NPs-free sLDMO/FAM/Cy5 was also studied.

Effector loading in photoactivatable DNA MO
As a proof-of-concept study, Dox was used as the model effector molecules and loaded within the photoactivatable DNA MO.To do this, 20 µL of psLDMO (0.

Photoactive release behaviors
To investigate the spatiotemporally controlled activity of the engineered DNA MO, their photothermal effects in response to NIR light were examined.Briefly, 100 µL of psLDMO aqueous solutions (0.005 μM, 0.01 μM, and 0.02 μM) were irradiated by an 808 nm NIR laser (2.0 W/cm 2 ) for 5 min, and the solution temperature was recorded with an IR thermal imaging system.Thereafter, 100 μL of psLDMO aqueous suspension (0.02 μM) were irradiated by an 808 nm laser at 2.0 W/cm 2 for 5 min, and the power was switched off.Such ON/OFF irradiation process was repeated five times, and the temperature changes of the psLDMO aqueous suspension in each cycle was determined in order to evaluate the photostability of psLDMO.
Next, the photoactive release dynamics of Dox from psLDMO/Dox was studied.
200 µL of psLDMO/Dox (0.02 μM) were irradiated by an 808 nm NIR laser at a 2.0 W/cm 2 power density for 5 min.The solution was centrifuged at 13000 rpm for 5 min, and the released Dox was quantified by measuring the fluorescence of Dox in the supernatant as Dox release percent (%) = released Dox/Dox encapsulated into psLDMO * 100% The above experiment was repeated 5 times, and the total released Dox in each cycle was calculated.As a negative control, the release of Dox from psLDMO/Dox in the dark within the same time period was also determined.

Cell lines and cell culture
4T1 cancer cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured in 1640 medium with 10% fetal bovine serum (FBS) (Zeta) and 1% penicillin-streptomycin (GIBCO) at 37 °C in a humid atmosphere with 5% CO2.

Cell internalization and photoactive drug release studies
To study cell internalization of these DNA MO, 10 5 4T1 cells per dish were seeded in confocal dishes and cultured for 24 h.The medium was removed, and a fresh medium containing psLDMO/Dox (5 µM equivalent of Dox) was added.Cells were then incubated for an additional 24 h.After washing with PBS twice, cells were irradiated by an 808 nm laser at 2.0 W/cm 2 for 5 min and cultured in fresh DMEM medium for 2 h.Cell internalization and Dox release were evaluated by CLSM.

Cell cytotoxicity
To evaluate the in vitro spatiotemporally controlled antitumor effect of these photoactivatable DNA coacervates, 4 T1 cells were seeded in wells of a 96-well plate (5 × 10 3 cells per well) and cultured for 24 h.Cells were then treated with psLDMO, psLDMO/Dox, and free Dox, respectively.The dosage of Dox was maintained at 5 µM, and the concentration of psLDMO was kept the same as that for the psLDMO and psLDMO/Dox groups.After 24-h incubation, cells were washed with PBS twice.
Thereafter, two groups of cells treated with psLDMO or psLDMO/Dox were irradiated by an 808 nm laser at 2.0 W/cm 2 for 5 min.After culture for an additional 24 h, cell viability of these groups was determined by the CCK-8 assay.

Western blot assay
4T1 cells treated with psLDMO or psLDMO/Dox in the absence or presence of laser irradiation were harvested and lysed with a lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40 substitute, 0.25% sodium deoxycholate, 1 mM sodium fluoride, 1 mM Na3VO4, and 1 mM EDTA), which was supplemented with protease inhibitor cocktail (Cell Signaling) and 1 mM phenylmethanesulfonyl fluoride.The protein concentration in each group was determined by a bicinchoninic acid (BCA) protein assay kit (Pierce/Thermo Scientific).Afterwards, 30 μg protein from each group were loaded on the SDS-PAGE gel and transferred to a PVDF membrane.The gel was then blocked with 5% skim milk in TBST (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween 20) and kept in a primary antibody solution at 4 ºC overnight.
After washing three times, the membrane was incubated in HRP-conjugated secondary antibody solution at room temperature for 1 h.Expression of the targeted protein was investigated by the enhanced chemiluminescence (ECL) detection system.

Pharmacokinetic and biodistribution studies
Cy5-lableld psLDMO was prepared for pharmacokinetic and biodistribution studies.At the indicated time intervals, mice were anesthetized with 2.5% isoflurane and imaged with an IVIS Lumina II imaging system (Caliper Life Science, USA).At 24 h post-injection, mice were sacrificed, and both tumor tissues and main organs (heart, liver, spleen, lung, and kidney) were collected for imaging.

In vivo spatiotemporally controlled antitumor studies
4T1 xenograft tumor-bearing mice were randomly divided into seven groups when the tumor size reached ~ 100 mm 3 and subjected to the following treatments: (i) PBS, (ii) PBS + laser, (iii) psLDMO, (iv) psLDMO + laser, (v), free Dox, (vi) psLDMO/Dox, and (vii) psLDMO/Dox + laser.All samples were injected through the tail vein.For groups (v) to (vii), the dosage of Dox was maintained at 5 mg/kg, and the concentration of psLDMO in groups (iii) and (vi) was kept the same as that for groups (v) to (vii).At 24 h post-injection, the mice in groups (ii), (iv) and (vii) were irradiated with an 808 nm laser with power density at 2 W/cm 2 for 5 min.Tumor temperature in each group was monitored.Afterwards, tumor size and body weight were monitored every day.
Tumor volume was calculated as Volume = length x width 2 /2.The relative volume of tumors was defined by normalizing the measured values to their initial sizes.Mice were finally sacrificed for histopathological analysis, including H&E, immunohistochemical, and immunofluorescence staining.

Tables
Table S1.DNA sequences for RCA.
3 µM) were mixed with 10 µM Dox dispersed in 100 µL of PBS for 24 h.Afterwards, the mixture was centrifuged and washed at 13000 rpm for 5 min.The supernatants were collected, and the amount of Dox in the supernatant was quantified by measuring fluorescence (Ex: 488 nm; Em: 590 nm).The loading efficiency of Dox in DNA MO was calculated as loading Dox = Total Dox -Dox in supernatant.

For
pharmacokinetic study, six-week-old BALB/c mice (n = 3) were injected with psLDMO/Cy5 through the tail vein at a dose of 2.0 nmol Cy5-DNA per mouse.At the indicated time points (0.15, 0.5, 1, 2, 4, 8, and 12 h), 20 µL of blood were collected from the retroorbital plexus of mouse eye and diluted to 100 µL with PBS containing 1 mg/mL heparin sulfate.The fluorescence intensity of blood was measured by using a Tecan spark-multimode microplate reader.For biodistribution study, 4T1 tumor-bearing BALB/c mice were established by subcutaneous implantation of 10 6 4T1 cells in PBS on the backside of six-week-old mice.Next, 200 μL of psLDMO/Cy5 in PBS were intravenously administered into 4T1 tumor-bearing BALB/c mice (n = 3) via the tail vein at 2.0 nmol Cy5-DNA per mouse.
Figure S1.Predicted secondary structures of the sequences used for RCA.

Figure S3 .
Figure S3.Confocal imaging of FAM-labeled mLDMO for fluorescence recovery after photobleaching analysis.

Figure S4 .
Figure S4.Quantitative analysis of the size of coacervates in Figure 2e.

Figure S5 .
Figure S5.(a) UV-vis spectrum and (b) TEM imagin of Pd NPs.(c) Size distribution statistics as calculated from panel b.

Figure S6 .
Figure S6.(a) Gel electrophoresis analysis of the stepwise formation of sLDMO with vs. without binding with Pd NPs.(b) Gel electrophoresis of the used primer and template DNA sequences in the absence vs. presence of Pd NPs.

Figure S8 .
Figure S8.TEM imaging and magnified TEM imaging of sLDMO after incubation with a low concentration of Pd NPs (0.125 ng/mL).

Figure S9 .
Figure S9.SEM imaging of sLDMO after incubation with Pd NPs at different concentrations.

Figure S10 .
Figure S10.SEM imaging of LLPS-driven formation of sLDMO with different RCA reaction durations, as well as corresponding fastener-bound condensation of sLDMO with Pd NPs.

Figure S12 .
Figure S12.Confocal imaging of Cy5-labeled pmLDMO and psLDMO after incubation in hypotonic PBS buffer for different time intervals.

Figure S13 .
Figure S13.Confocal imaging of FAM-labeled psLDMO after incubation in 10% FBS buffer for different time intervals.

Figure S14 .
Figure S14.Quantitative analysis of confocal images in Figure S13.

Figure S15 .
Figure S15.Confocal imaging of FAM-labeled psLDMO for fluorescence recovery after photobleaching analysis.

Figure S17 .
Figure S17.TEM imaging of psLDMO obtained after 24-h RCA before and after binding with Pd NPs.

Figure S25 .
Figure S25.Time-dependent release of Dox from psLDMO/Dox suspended in the 10%

Figure S28 .
Figure S28.(a) Fluorescence imaging of tumor tissues and main organs harvested from

Figure S31 .
Figure S31.Fluorescence imaging of tumor tissues and main organs harvested from psLDMO/Dox-treated mice at 24 h post injection.

Figure S34 .
Figure S34.Individual tumor growth curves of 4T1 tumor-bearing mice after different treatments.

Figure S35 .
Figure S35.Tumor weights of 4T1 tumor-bearing mice harvested from different treatment groups.Data were presented as mean values ± S.D.

Table S2 .
ICP-OES analysis of palladium in sLDMO and psLDMO.

Table S3 .
ICP-OES analysis of intracellular palladium after incubation with or