Synthetic Condensates and Cell-Like Architectures from Amphiphilic DNA Nanostructures

Summary

We present a protocol for preparing synthetic biomolecular condensates consisting of amphiphilic DNA nanostars starting from their constituent DNA oligonucleotides. Condensates are produced from either a single nanostar component or two components and are modified to sustain in vitro transcription of RNA from an embedded DNA template.

Abstract

Synthetic droplets and condensates are becoming increasingly common constituents of advanced biomimetic systems and synthetic cells, where they can be used to establish compartmentalization and sustain life-like responses. Synthetic DNA nanostructures have demonstrated significant potential as condensate-forming building blocks owing to their programmable shape, chemical functionalization, and self-assembly behavior. We have recently demonstrated that amphiphilic DNA "nanostars", obtained by labeling DNA junctions with hydrophobic moieties, constitute a particularly robust and versatile solution. The resulting amphiphilic DNA condensates can be programmed to display complex, multi-compartment internal architectures, structurally respond to various external stimuli, synthesize macromolecules, capture and release payloads, undergo morphological transformations, and interact with live cells. Here, we demonstrate protocols for preparing amphiphilic DNA condensates starting from constituent DNA oligonucleotides. We will address (i) single-component systems forming uniform condensates, (ii) two-component systems forming core-shell condensates, and (iii) systems in which the condensates are modified to support in vitro transcription of RNA nanostructures.

Introduction

Synthetic cells are micrometer-scale (10-50 µm) devices constructed from the bottom-up to replicate functions and structures of extant biological cells^1,2. Synthetic cells are often bound by membranes constructed from lipid bilayer vesicles^3,4,5,6,7, polymersomes^8,9, or proteinosomes^10,11, which can also be....

Protocol

NOTE: The protocol is divided into three sections. Section 1 describes the prerequisite steps, including the preparation of DNA oligonucleotides and glass capillaries. Section 2 describes the preparation of C-star condensates of various designs, including one- and two-component designs, and their extraction from the glass capillaries. Section 3 describes the use of one-component RNA templating C-star condensates for the synthesis of an RNA aptamer. The user must follow good lab practice throughout, ensure that all necess.......

Discussion

The protocol described here provides an approach for the preparation of one- or two- component condensates from amphiphilic DNA nanostars, with design variations to introduce different responses into the condensates. The given protocol produces condensates in a buffer solution of 0.3 M NaCl in TE, but the buffer conditions can be amended by appropriately modifying the volumes listed above. Previous work has studied the formation of C-star condensates in 0.2 M NaCl in TE and 0.1 M NaCl in TE and in phosphate-buffered sali.......

Materials

Name	Company	Catalog Number	Comments
0.22 μm syringe filters	Sigma-Aldrich	SLGVR33RB
24 x 60 mm #1.5 Rectangular cover glasses, Menzel Gläser	VWR	631-0853
2-Propanol	Sigma-Aldrich	34683
6 L Ultrasonic Cleaner with Digital Timer and Heat, 230 VAC	Cole-Parmer	WZ-08895-11
Araldite Rapid Adhesive 2 Part Epoxy Glue	RS	ARA-400005
Bio-Rad C1000 thermal cycler	Bio-Rad	1851197
Brand Microcentrifuge Tube 2 mL with Locking Lid	Fisher Scientific	15338665	2 mL microcentrifuge tubes for the extraction of C-star condensates
Diamond Scribing Pen	RS	394-217
Difluoro-4-hydroxybenzylidene imidazolidinone (DFHBI)	Sigma-Aldrich	SML1627
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	472301
Eppendorf PCR Clean Colorless Safe-Lock Centrifuge Tubes	Fisher Scientific	0030123301	0.5 mL microcentrifuge tubes for the preparation of C-star mixtures
Ethanol Absolute 99.8+%	Fisher Scientific	10437341	70% ethanol is sufficient for cleaning purposes
Fisherbrand ZX4 IR Vortex Mixer	Fisherbrand	13284769
Hellmanex III	Hellma	9-307-011-4-507
Hollow Rectangle Capillaries ID 0.40 x 4.00 mm, 50 mm in length	CM Scientific	2540-50
Mineral oil	Sigma-Aldrich	69794
Mini Centrifuge, 230 V	PRISM(TM)	Z763128
NaCl	Sigma-Aldrich	S3014
NanoDrop One Spectrophotometer	Thermo Fisher Scientific	ND-ONE-W	Used to measure absorbance of oligonucleotides for concentration calculations
Oligonucleotides	Integrated DNA Technologies	Custom	Oligonucleotide sequences are unique to the C-star design required.
ScriptGuard RNase inhibitor	CELLSCRIPT	C-SRI6310K	RNase inhibitor
T7-FlashScribe Transcription Kit	Cambio	C-ASF3507
Tris-EDTA buffer, 100x stock solution	Sigma-Aldrich	574793
UltraPure DNase/RNase-Free Distilled Water	Invitrogen	10977035
VWR Spec-Wipe 3 Wipers	VWR	21914-758