Determination of In Vitro and Cellular Turn-on Kinetics for Fluorogenic RNA Aptamers

Summary

The protocol presents two methods to determine the kinetics of the fluorogenic RNA aptamers Spinach2 and Broccoli. The first method describes how to measure fluorogenic aptamer kinetics in vitro with a plate reader, while the second method details the measurement of fluorogenic aptamer kinetics in cells by flow cytometry.

Abstract

Fluorogenic RNA aptamers have been applied in live cells to tag and visualize RNAs, report on gene expression, and activate fluorescent biosensors that detect levels of metabolites and signaling molecules. In order to study dynamic changes in each of these systems, it is desirable to obtain real-time measurements, but the accuracy of the measurements depends on the kinetics of the fluorogenic reaction being faster than the sampling frequency. Here, we describe methods to determine the in vitro and cellular turn-on kinetics for fluorogenic RNA aptamers using a plate reader equipped with a sample injector and a flow cytometer, respectively. We show that the in vitro kinetics for the fluorescence activation of the Spinach2 and Broccoli aptamers can be modeled as two-phase association reactions and have differing fast phase rate constants of 0.56 s⁻¹ and 0.35 s⁻¹, respectively. In addition, we show that the cellular kinetics for the fluorescence activation of Spinach2 in Escherichia coli, which is further limited by dye diffusion into the Gram-negative bacteria, is still sufficiently rapid to enable accurate sampling frequency on the minute timescale. These methods to analyze fluorescence activation kinetics are applicable to other fluorogenic RNA aptamers that have been developed.

Introduction

Fluorogenic reactions are chemical reactions that generate a fluorescence signal. Fluorogenic RNA aptamers typically perform this function by binding a small molecule dye to enhance its fluorescence quantum yield (Figure 1A)¹. Different fluorogenic RNA aptamer systems have been developed and consist of specific RNA aptamer sequences and the corresponding dye ligands¹. Fluorogenic RNA aptamers have been appended to RNA transcripts as fluorescent tags that enable live cell imaging of mRNAs and non-coding RNAs^2,3,

Protocol

1. In vitro kinetics experiment

1. Preparation of DNA templates by PCR
   1. Set up PCR reaction(s): To prepare PCR reactions, combine the following reagents in a thin-walled PCR tube:
      33 µL of double-distilled water (ddH₂O)
      10 µL of 5x buffer for high-fidelity DNA polymerase
      5 µL of 2 mM each deoxyribonucleoside triphosphate (dNTP)
      0.5 µL of 40 µM forward primer
      0.5 µL of 40 µM reverse primer

Discussion

For the in vitro kinetics experiment, the same general protocol can be modified to measure the in vitro kinetics of an RNA-based fluorescent biosensor containing both a ligand-binding and fluorophore-binding domain⁸. In this case, the RNA should be incubated with the fluorophore prior to measurements upon injecting the ligand in order to obtain ligand response kinetics. If high variability is observed between the replicates, one can troubleshoot by checking that each sample is al.......

Materials

Name	Company	Catalog Number	Comments
Agarose	Thermo Fischer Scientific	BP160500
Agarose gel electrophoresis equipment	Thermo Fischer Scientific	B1A-BP
Alpha D-(+)-lactose monohydrate	Thermo Fischer Scientific	18-600-440
Amber 1.5 mL microcentrifuge tubes	Thermo Fischer Scientific	22431021
Ammonium persulfate (APS)	Sigma-Aldrich	A3678
Ammonium sulfate ((NH4)2SO4)	Sigma-Aldrich	A4418
Attune NxT Flow cytometer	Thermo Fischer Scientific	A24861
Attune 1x Focusing Fluid	Thermo Fischer Scientific	A24904
Attune Shutdown Solution	Thermo Fischer Scientific	A24975
Attune Performance Tracking Beads	Thermo Fischer Scientific	4449754
Attune Wash Solution	Thermo Fischer Scientific	J24974
Boric acid	Sigma-Aldrich	B6768
Bromophenol blue	Sigma-Aldrich	B0126
Carbenicillin disodium salt	Sigma-Aldrich	C3416
Chlorine Bleach	Amazon	B07J6FJR8D
Corning Costar 96-well plate	Daigger Scientific	EF86610A
Culture Tubes, 12 mm x 75 mm, 5 mL with attached dual position cap	Globe Scientific	05-402-31
DFHBI	Sigma-Aldrich	SML1627
DFHBI-1T	Sigma-Aldrich	SML2697
D-Glucose (anhydrous)	Acros Organics	AC410955000
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	D8418
Dithiothreitol (DTT)	Sigma-Aldrich	DTT-RO
DNA loading dye	New England Biolabs	B7025S
DNA LoBind Tubes (2.0 mL)	Eppendorf	22431048
dNTPs: dATP, dCTP, dGTP, dTTP	New England Biolabs	N0446S
EDTA, pH 8.0	Gibco, Life Technologies	AM9260G
Ethanol (EtOH)	Sigma-Aldrich	E7023
Filter-tip micropipettor tips	Thermo Fischer Scientific	AM12635, AM12648, AM12655, AM12665
FlowJo Software	BD Biosciences	N/A	FlowJo v10 Software
Fluorescent plate reader with heating control	VWR	10014-924
Gel electrophoresis power supply	Thermo Fischer Scientific	EC3000XL2
Glycerol	Sigma-Aldrich	G5516
Glycogen AM95010	Thermo Fischer Scientific	AM95010
GraphPad Prism	Dotmatics	N/A	Analysis software from Academic Group License
Heat block	Thomas Scientific	1159Z11
HEPES	Sigma-Aldrich	H-4034
Inorganic pyrophosphatase	Sigma-Aldrich	I1643-500UN
Low Molecular Weight DNA Ladder	New England Biolabs	N3233L	Supplied with free vial of Gel Loading Dye, Purple (6x), no SDS (NEB #B7025).
Magnesium chloride hexahydrate (MgCl2)	Sigma-Aldrich	M2670
Magnesium sulfate (MgSO4)	Fisher Scientific	MFCD00011110
Microcentrifuge tubes (1.5 mL)	Eppendorf	22363204
Microcentrifuge with temperature control	Marshall Scientific	EP-5415R
Micropipettors	Gilson	FA10001M, FA10003M, FA10005M, FA10006M
Micropipettor tips	Sigma-Aldrich	Z369004, AXYT200CR, AXYT1000CR
Millipore water filter with BioPak unit	Sigma-Aldrich	CDUFBI001, ZRQSVR3WW
Narrow micropipettor pipette tips	DOT Scientific	RN005R-LRS
PBS, 10x	Thermo Fischer Scientific	BP39920
PCR clean-up kit	Qiagen	28181
PCR primers and templates	Integrated DNA technologies
PCR thermocycler for thin-walled PCR tubes	Bio-Rad	1851148
PCR thermocycler for 0.5 mL tubes	Techne	5PRIME/C
pET31b-T7-Spinach2 Plasmid	Addgene	Plasmid #79783
Phusion High-Fidelity DNA polymerase	New England Biolabs	M0530L	Purchase of Phusion High-Fideldity Enzyme is supplied with 5x Phusion HF Buffer, 5x Phusion GC Buffer, and MgCl2 and DMSO solutions.
Polyacrylamide gel electrophoresis gel comb, C.B.S. Scientific	C.B.S. Scientific	VGC-1508
Polyacrylamide gel electrophoresis equipment	C.B.S. Scientific	ASG-250
Potassium chloride (KCl)	Sigma-Aldrich	P9333
Potassium phosphate monobasic	Sigma-Aldrich	P5655
Razor blades	Genesee Scientific	38-101
rNTPs: ATP, CTP, GTP, UTP	New England Biolabs	N0450L
SDS	Sigma-Aldrich	L3771
Short wave UV light source	Thermo Fischer Scientific	11758221
Sodium carbonate (Na2CO3)	Sigma-Aldrich	S7795
Sodium chloride (NaCl)	Sigma-Aldrich	S7653
Sodium hydroxide (NaOH)	Sigma-Aldrich	S8045
Sodium phosphate dibasic, anhydrous	Thermo Fischer Scientific	S375-500
SoftMax Pro	Molecular Devices	N/A	SoftMax Pro 6.5.1 (platereader software) obtained through Academic Group License
Sterile filter units	Thermo Fischer Scientific	09-741-88
Sucrose	Sigma-Aldrich	S0389
SYBR Safe DNA gel stain	Thermo Fischer Scientific	S33102
TAE buffer for agarose gel electrophoresis	Thermo Fischer Scientific	AM9869
Tetramethylethylenediamine (TEMED)	Sigma-Aldrich	T9281
Tris base	Sigma-Aldrich	TRIS-RO
Tryptone (granulated)	Thermo Fischer Scientific	M0251S
T7 RNA polymerase	New England Biolabs	M0251S
Urea-PAGE Gel system	National Diagnostics	EC-833
UV fluorescent TLC plate	Sigma-Aldrich	1.05789.0001
UV/Vis spectrophotometer	Thermo Fischer Scientific	ND-8000-GL
Vortex mixer	Thermo Fischer Scientific	2215415
Xylene cyanol	Sigma-Aldrich	X4126
Yeast Extract (Granulated)	Thermo Fischer Scientific	BP9727-2