Preparation of Multifunctional Silk-Based Microcapsules Loaded with DNA Plasmids Encoding RNA Aptamers and Riboswitches

Summary

The protocol describes the formation of robust and biocompatible DNA-laden microcapsules as multiplexed in vitro biosensors capable of tracking several ligands.

Abstract

We introduce a protocol for the preparation of DNA-laden silk fibroin microcapsules via the Layer-by-Layer (LbL) assembly method on sacrificial spherical cores. Following adsorption of a prime layer and DNA plasmids, the formation of robust microcapsules was facilitated by inducing β-sheets in silk secondary structure during acute dehydration of a single silk layer. Hence, the layering occurred via multiple hydrogen bonding and hydrophobic interactions. Upon adsorption of multilayered shells, the core-shell structures can be further functionalized with gold nanoparticles (AuNPs) and/or antibodies (IgG) to be used for remote sensing and/or targeted delivery. Adjusting several key parameters during sequential deposition of key macromolecules on silica cores such as the presence of a polymer primer, the concentration of DNA and silk protein, as well as a number of adsorbed layers resulted in biocompatible, DNA-laden microcapsules with variable permeability and DNA loadings. Upon dissolution of silica cores, the protocol demonstrated the formation of hollow and robust microcapsules with DNA plasmids immobilized to the inner surface of the capsule membrane. Creating a selectively permeable biocompatible membrane between the DNA plasmids and the external environment preserved the DNA during long-term storage and played an important role in the improved output response from spatially confined plasmids. The activity of DNA templates and their accessibility were tested during in vitro transcription and translation reactions (cell-free systems). DNA plasmids encoding RNA light-up aptamers and riboswitches were successfully activated with corresponding analytes, as was visualized during localization of fluorescently labeled RNA transcripts or GFPa1 protein in the shell membranes.

Introduction

The field of synthetic biology offers unique opportunities to develop sensing capabilities by exploiting natural mechanisms evolved by microorganisms to monitor their environment and potential threats. Importantly, these sensing mechanisms are typically linked to a response that protects these microorganisms from harmful exposure, regulating gene expression to mitigate negative effects or prevent intake of toxic materials. There have been significant efforts to engineer these microorganisms to create whole-cell sensors taking advantage of these natural responses but re-directing them to recognize novel targets and/or to produce a measurable signal that can be measured....

Protocol

1. Construction of plasmid vector.

1. Construct a plasmid vector (pSALv-RS-GFPa1, 3.4 kb) by amplification of the coding sequence of a theophylline riboswitch (ThyRS) coupled with GFPa1 from pJ201:23976-RS-GFPa1 vector (designed and created by DNA2.0) and insertion into E. coli expression vector, pSAL¹³. Use forward (5'-CGTGGTACCGGTGATACCAGCATCGTCTTGATG-3') and reverse (5'-CGTGCTCAGCTTAAGCCAGCTCGTAG-3') primers to amplify the coding sequence of ThyRS coupled with GFPa1 and perform a PCR reaction in 50 µL volume using DNA polymerase according to the manufacturer's protocol¹⁴.

Results

Here, the study addresses the functionality of DNA templates encoding different sensor designs (two types of RNA-regulated transcription/translation elements) after encapsulation in silk protein capsules. Microcapsules were prepared via templated Layer-by-Layer (LbL) assembly of the key components: A prime layer, DNA plasmids encoding sensor designs, and silk fibroin biopolymer (Figure 2). Deposition of macromolecules in a layered fashion allows controlling the permeability of the capsule me.......

Discussion

Selectively permeable hydrogel microcapsules loaded with various types of DNA-encoded sensor designs can be prepared following this protocol. One of the distinctive features of the LbL approach is the ability to tailor the complexity of microcapsules during the bottom-up assembly, which usually starts with the adsorption of molecular species on sacrificial templates. By carefully adjusting concentrations of the initial components, pH conditions, and the number of layers, microcapsules with different DNA loading parameter.......

Materials

Name	Company	Catalog Number	Comments
(Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-1-(2,2,2-trifluoroethyl)-1H-imidazol-5(4 H)-one (DFHBI-1T)	Lucerna	DFHBI-1T
5x T4 DNA Ligase Buffer	ThermoFisher Scientific	46300-018
6x Blue Gel Loading Dye	New England BioLabs	B7021S
96-well plates, black circular	Corning	3601
Agarose	Sigma-Aldrich	A9539	BioReagent, for molecular biology, low EEO
Ampicillin sodium salt	Sigma-Aldrich	A0166	powder or crystals, BioReagent, suitable for cell culture
BlpI restriction enzymes	New England BioLabs	R0585S
Corning Disposable Vacuum Filter/Storage Systems	FisherScientific	09-761-1
Dimethyl sulfoxide, DMSO	Sigma-Aldrich	472301	ACS reagent, ≥99.9%
DNA Plasmid, pET28c-F30-2x Broccoli (5.4 kb), BrocApt.	Addgene	Plasmid #66788
DyLightTM550 Antibody Labeling kit (Invitrogen)	ThermoFisher Scientific	84530
E. coli S30 extract system for circular DNA	Promega	L1020
Falcon Conical centrifuge tubes, 15 mL	FisherScientific	14-959-53A
Falcon Conical centrifuge tubes, 50 mL		14-432-22
Fisherbrand Microcentrifuge tubes, 1.5 mL	FisherScientific	05-408-129
Hydrofluoric acid, HF	Sigma-Aldrich	695068	ACS reagent, 48%
Kanamycin sulfate	Sigma-Aldrich	60615	mixture of Kanamycin A (main component) and Kanamycin B and C
KpnI restriction enzymes	New England BioLabs	R0142S
LB agar plate supplemented with 100 µg/mL ampicillin	Sigma-Aldrich	L5667	pre-poured agar plates with 100 µg/mL ampicillin
LB agar plate supplemented with 50 µg/mL kanamycin	Sigma-Aldrich	L0543	pre-poured agar plates with 50 µg/mL kanamycin
LB broth (Lennox grade)	Sigma-Aldrich	L3022
Lithium bromide, LiBr	Sigma-Aldrich	213225	ReagentPlus, ≥99%
Max Efficiency DH5-α competent E. coli strain	ThermoFisher Scientific	18258012
Methanol	MilliporeSigma	322415	anhydrous, 99.8%
MilliQ-water	EMD MilliPore		Milli-Q Reference Water Purification System
MinElute PCR Purification Kit	Qiagen	28004
N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, EDC	Sigma-Aldrich	E1769
PBS (phosphate buffered saline)	ThermoFisher Scientific	10010023	1x PBS, pH 7.4
Phusion High-Fidelity DNA Polymerase	New England Biolabs	M0530S
Polyethylenimine, branched	Sigma-Aldrich	408727	average Mw ~25,000
PURExpress In Vitro Protein Synthesis Kit	New England BioLabs	E6800S
QIAEX II Gel Extraction Kit	Qiagen	20021
QIAprep Spin Miniprep Kit	Qiagen	27104
Quick-Load 2-Log DNA Ladder (0.1-10.0 kb)	New England BioLabs	N0469S
SiO₂ silica microspheres, 4.0 µm	Polysciences, Inc.	24331-15	10% aqueous solution
Slide-A-Lyzer G2 Dialysis Cassettes, 3.5K MWCO, 15 mL	ThermoFisher Scientific	87724
Sodium carbonate, Na₂CO₃	Sigma-Aldrich	222321	ACS reagent, anhydrous, ≥99.5%, powder
Spectrum Spectra/Por Float-A-Lyzer G2 Dialysis Devices	FisherScientific	08-607-008	Spectrum G235058
SYBR Safe DNA gel stain	ThermoFisher Scientific	S33102
T4 DNA Ligase (5 U/µL)	ThermoFisher Scientific	EL0011
Theophylline	Sigma-Aldrich	T1633	anhydrous, ≥99%, powder
Tris Acetate-EDTA buffer (TAE buffer)	Sigma-Aldrich	T6025	Contains 40 mM Tris-acetate and 1 mM EDTA, pH 8.3.
UltraPure DNase/RNase-Free Distilled Water	FisherScientific	10-977-023
ZymoPURE II Plasmid MaxiPrep kit	ZymoResearch	D4202