Fluorescent Visualization of Mango-tagged RNA in Polyacrylamide Gels via a Poststaining Method

Summary

Here we present a sensitive, rapid, and discriminating post-gel staining method to image RNAs tagged with RNA Mango aptamers I, II, III, or IV, using either native or denaturing polyacrylamide gel electrophoresis (PAGE) gels. After running standard PAGE gels, Mango-tagged RNA can be easily stained with TO1-Biotin and then analyzed using commonly available fluorescence readers.

Abstract

Native and denaturing polyacrylamide gels are routinely used to characterize ribonucleoprotein (RNP) complex mobility and to measure RNA size, respectively. As many gel-imaging techniques use nonspecific stains or expensive fluorophore probes, sensitive, discriminating, and economical gel-imaging methodologies are highly desirable. RNA Mango core sequences are small (19–22 nt) sequence motifs that, when closed by an arbitrary RNA stem, can be simply and inexpensively appended to an RNA of interest. These Mango tags bind with high affinity and specificity to a thiazole-orange fluorophore ligand called TO1-Biotin, which becomes thousands of times more fluorescent upon binding. Here we show that Mango I, II, III, and IV can be used to specifically image RNA in gels with high sensitivity. As little as 62.5 fmol of RNA in native gels and 125 fmol of RNA in denaturing gels can be detected by soaking gels in an imaging buffer containing potassium and 20 nM TO1-Biotin for 30 min. We demonstrate the specificity of the Mango-tagged system by imaging a Mango-tagged 6S bacterial RNA in the context of a complex mixture of total bacterial RNA.

Introduction

Mango is an RNA tagging system consisting of a set of four small fluorescent RNA aptamers that bind tightly (nanomolar binding) to simple derivatives of the thiazole-orange (TO1-Biotin, Figure 1A)^1,2,3. Upon binding, the fluorescence of this ligand is increased 1,000- to 4,000-fold depending on the specific aptamer. The high brightness of the Mango system, which for Mango III exceeds that of enhanced green fluorescent protein (eGFP), combined with the nanomolar binding affinity of the RNA Mango aptamers, allows it to be used both in the imaging a....

Protocol

1. Preparation of the reagents

1. TO1-Biotin poststaining solution (gel staining solution)
   1. Make 1 L of 1 M phosphate buffer at pH 7.2 at 25 °C by adding 342 mL of 1 M of Na₂HPO₄ and 158 mL of 1 M NaH₂PO₄. Adjust pH to 7.2 at 50 mM by adding the appropriate phosphate solution. Sterile-filter using a 0.2 µm filter and store the solution in plasticware at room temperature.
   2. Prepare 5x gel staining solution (without TO1-Biotin) as follows. Make up a 1 L solution by mixing 247.5 mL of ddH₂O, 700 mL of 1 M KCl, 50 mL of 1 M phosphate buffer (pH 7.2), and 2.5 mL o....

Results

Short Mango-tagged RNAs were prepared as described in the protocol section. Assuming that fluorescence in denaturing conditions would be most difficult to observe owing to the presence of urea in the gels, we first studied the resistance of the Mango aptamers to urea, which acts as a nucleic acid denaturant. We found that Mango aptamers are substantially resistant to denaturation up to a urea concentration of approximately 1 M (Figure 2A). Prior to adding gel staining sol.......

Discussion

A significant advantage of the Mango fluorescent tag is that a single tag can be used in multiple ways. The high brightness and affinity of these aptamers make them useful not only for in cell visualization² but also for in vitro RNA or RNP purification⁴. Therefore, gel imaging extends the versatility of the Mango tag in a straightforward way. Mango gel imaging sensitivity is slightly less than that of a northern blot¹⁴ but can easily detect 60–.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.8mm Thick Comb 14 Wells for 30 mL PAGE gels	LabRepCo	11956042
101-1000 µL  tips	Fisher	02-707-511
20-200 µL low retention  tips	Fisher Scientific	02-717-143
Acrylamide:N,N'-methylenebisacrylamide (40% 19:1)	Bioreagents	BP1406-1	Acute toxicity
Acrylamide:N,N'-methylenebisacrylamide (40% 29:1)	Fisher	BP1408-1	Acute toxicity
Agar	Anachemia	02116-380
Aluminium backed TLC plate	Sigma-Aldrich	1164840001
Amersham Imager 600	GE Healthcare Lifesciences	29083461
Ammonium Persulfate	Biorad	161-0700	Harmful
BL21 cells	NEB	C2527H
Boric Acid	ACP	B-2940
Bromophenol Blue sodium salt	Sigma	B8026-25G
Chloloform	ACP	C3300
Dithiothreitol	Sigma Aldrich Alcohols	D0632-5G
DNase I	ThermoFisher	EN0525
EDTA Disodium Salt	ACP	E-4320
Ethanol	Commerial	P016EAAN
Flat Gel Loading tips	Costar	CS004854
Formamide 99%	Alfa Aesar	A11076
Gel apparatus set with spacers and combs	LabRepCo	41077017
Glass Dish with Plastic lid	Pyrex	1122963	Should be large enough to fit your gel piece
Glycerol	Anachemia	43567-540
HCl	Anachemia	464140468
ImageQuanTL	GE Healthcare Lifesciences	29000605
IPTG	Invitrogen	15529-019
KCl	ACP	P-2940
MgCl2	Caledron	4903-01
MgSO4	Sigma-Aldrich	M3409
NaCl	ACP	S-2830
NaOH	BDH	BDH9292
Orbital Rotator	Lab-Line
Phenol	Invitrogen	15513-039
Round Gel Loading tips	Costar	CS004853
Sodium Phosphate dibasic	Caledron	8120-1
Sodium Phosphate monobasic	Caledron	8180-01
SYBRGold	ThermoFisher	S11494
T7 RNA Polymerase	ABM	E041
TEMED	Sigma-Aldrich	T7024-50 ml
TO1-3PEG-Biotin Fluorophore	ABM	G955
Tris Base	Fisher	BP152-500
Tryptone	Fisher	BP1421-500
Tween-20	Sigma	P9496-100
Urea	Fisher	U15-3
Xylene Cyanol	Sigma	X4126-10G
Yeast Extract	Bioshop	YEX401.500