MitoDNA™ Red: A Powerful Tool for Labeling and Detection of mtDNA

Abstract

Mitochondria are the only organelles in a cell that carry DNA apart from the nucleus. Mitochondrial DNA (mtDNA) plays a key role in cellular function, development, and disease. It contains the genetic information for synthesizing enzymes, transfer RNA (tRNA), and ribosomal RNA (rRNA). Mutations and disorders in mtDNA can lead to a range of health issues, including age-related hearing loss, diabetes, and failures in the brain, heart, and liver. Effective visualization and quantification of mtDNA are essential for understanding mitochondrial dynamics and investigating mitochondrial-related diseases. This article introduces advanced techniques for labeling and detecting mtDNA using novel fluorescent dyes, MitoDNA™ Red, with a focus on microscopy methods.

Introduction

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Mitochondrial DNA (mtDNA) refers to the extrachromosomal DNA found specifically within the mitochondria, rather than in other parts of the cell. mtDNA encodes numerous proteins, peptides, transfer RNAs and ribosomal RNA that support various physiological cellular processes like development, disease progression, and aging. Mutations and disorders in mtDNA can lead to a range of health issues, including age-related hearing loss, diabetes, and failures in the brain, heart, and liver. Such mtDNA-linked diseases depend not only on the specific mutation present but also on the degree of heteroplasmy. Detection of mitochondrial DNA is limited by the availability of very few probes which can specifically detect mtDNA. Commonly used fluorescent DNA probes, such as DAPI, Hoechst, or SYBR® Green, lack the specificity required for mitochondrial targeting, primarily staining nuclear DNA.

Fluorescent dyes are widely used for mtDNA labeling due to their simple protocols, minimal hands-on steps, and real-time imaging capabilities in live cells. Using fluorescent dyes to label and monitor mtDNA without the need for fixing and permeabilizing the cells, allows for the possibility of additional tests that maybe needed in the study. MitoDNA™ Red 610, MitoDNA™ Red 680, and MitoDNA Red 710™ are cell-permeable dyes that specifically stain mtDNA in live cells, offering an efficient method for the dynamic imaging of mtDNA. These dyes exhibit a large Stokes Shift, providing an excellent signal-to-noise ratio and enabling easy multiplex staining with other fluorescent probes. Fluorescent microscopy has proven to be a powerful tool in studying mitochondrial dynamics, offering exceptionally high-resolution images, and is arguably one of the easiest visualization techniques to perform. Other microscopy techniques like confocal, super-resolution, light-sheet, cryo-tomography, and electron microscopy can also be employed for detecting fluorescent mtDNA.

Materials and Methods

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Cell Culture

HeLa cells were cultured and maintained in DMEM with 10% Fetal Bovine Serum (FBS) and 1% antibiotic (100 U/mL Penicillin and 100 μg/mL Streptomycin), under a humidified atmosphere at 37°C and 5% CO2.

MtDNA staining in live cells

HeLa cells were seeded overnight in 96-well clear-bottom dark plates (~80-90% confluency). 10 mM stock solution in DMSO for MitoDNA™ Red 610/680/710 (AAT Bioquest #22687, #22688, #22689) was prepared and further diluted to a 5μM working solution by diluting the stock solution in Hanks' solution with 20 mM HEPES buffer (HHBS). Cell culture medium was removed and 100 μL of MitoDNA™ Red working solution was added directly to the cells and incubated at 37°C for 10 minutes, protected from light. The dye working solution was removed and cells were washed twice with HHBS buffer. To check whether the staining is specific for DNA, DNase enzyme was added to stained cells and incubated 1 hour at 37°C. Stained cells were analyzed using a fluorescence microscope with excitation/emission settings of 490/610 nm for MitoDNA™ Red 610, 600/680 nm for MitoDNA™ Red 680, and 510/710 nm for MitoDNA™ Red 710.

Mitochondrial Co-localization Studies

HeLa cells were seeded overnight in 96-well clear-bottom dark plates (~80-90% confluency). 10 mM stock solution was prepared in DMSO for MitoDNA™ Red 610. 5μM working solution was prepared by diluting the stock solution in Hanks' solution with 20 mM HEPES buffer (HHBS) along with MitoLite™ NIR FX690 (AAT Bioquest #22690). The culture medium was removed and 100 μL of dye working solution was added directly to the cells and incubated at 37°C for 5-15 minutes, protected from light. The dye working solution was removed and cells were washed twice with HHBS buffer. To check whether the staining is specific for DNA, DNase enzyme was added to stained cells and incubated 1 hour at 37°C. Co-stained cells were analyzed using a fluorescence microscope with excitation/emission settings of 490/610 nm for MitoDNA™ Red 610 and 658/691nm for MitoLite™ NIR FX690.

Results

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Specificity test for MtDNA staining in live cells

MitoDNA™ Red 610/680/710 dyes (5-10 μM) in HHBS buffer were added to the overnight seeded HeLa cells and fluorescence was monitored by fluorescence microscope. Cells were then incubated with DNase and monitored again to understand the effect of DNase on MitoDNA dyes. All three dyes gave specific fluorescence in live cells. No nuclear staining was seen. The fluorescence signal is lost upon DNase treatment due to digestion of DNA. This data suggests that these dyes have a specificity for mtDNA.

Mitochondrial co-localization with MitoLite™ NIR FX690

Overnight seeded HeLa cells were co-incubated with MitoDNA™ Red 610 dye and a known mitochondrial labeling dye, MitoLite™ NIR FX690 in HHBS buffer and fluorescence was monitored by fluorescence microscope (Using two channels to generate overlay of both staining). Cells were then incubated with DNase and monitored again to understand the effect of DNase on MitoDNA™ dyes. Both dyes gave overlapping signals in live cells indicating that the MitoDNA™ Red 610 dye is staining mitochondria. No nuclear staining was seen. The fluorescence signal of MitoDNA™ Red 610 dye is lost upon DNase treatment due to digestion of DNA, while the signal from MitoLite™ NIR FX690 was still intact. This suggests that the MitoDNA™ Red 610 dye has a specificity for mitochondrial DNA. Co-localization studies were performed with other two MitoDNA™ dyes as well (Data is not shown here).

Discussion

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MitoDNA™ Red: A Powerful Tool for Fluorescent Labeling and Detection of mtDNA

Although a wide variety of DNA binding dyes exist for labeling the nuclear DNA, only a handful have shown to be able to label mtDNA within the mitochondria. This is because mtDNA binding dyes must traverse not just the plasma membrane, but also the mitochondrial outer and inner membranes to gain access into the mitochondrial matrix and mtDNA. Traditionally used DNA dyes lack the specificity when targeting mtDNA and tend to stain nuclear DNA. Novel fluorescent mtDNA dyes, like MitoDNA™ Red 610, MitoDNA™ Red 680, and MitoDNA™ Red 710, as shown here have proven more effective to precisely target mtDNA within a cell sample. MitoDNA™ Red dyes are highly cell-permeable dye that selectively stains mtDNA in live cells, providing a method for dynamic imaging of mtDNA. These dyes offer a high signal-to-noise ratio and enable multiplex staining with other fluorescent probes. Additionally, MitoDNA™ Red produces a relatively bright and photostable signal. The versatile applications of MitoDNA™ Red dyes using a range of imaging techniques, provides researchers with powerful tool for studying mitochondrial DNA dynamics.

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