Communication pubs.acs.org/JACS
Creation of a Synthetic Ligand for Mitochondrial DNA Sequence Recognition and Promoter-Specific Transcription Suppression Takuya Hidaka,† Ganesh N. Pandian,*,‡ Junichi Taniguchi,† Tomohiro Nobeyama,§ Kaori Hashiya,† Toshikazu Bando,† and Hiroshi Sugiyama*,†,‡ †
Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo, Kyoto 606-8502, Japan Institute for Integrated Cell-Material Science (WPI-iCeMS), Kyoto University, Sakyo, Kyoto 606-8501, Japan § Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, Imizu, Toyama 939-0398, Japan ‡
S Supporting Information *
mitochondrial transcription factor A (TFAM), and mitochondrial transcription factor B2 (TFB2M).5−7 Programmable naturally occurring proteins have been used to edit the mitochondrial genome.8,9 Nevertheless, no synthetic ligands have been developed that are adept at (1) mitochondrial localization, (2) DNA recognition, and (3) targeted suppression of endogenous gene expression. N-Methylpyrrole-N-methylimidazole polyamides (PIPs) bind to the minor groove of double-strand DNA in a sequencespecific manner. Antiparallel pyrrole/imidazole pairs recognize C/G base pairs, and pyrrole/pyrrole pairs recognize A/T or T/ A base pairs.10 PIPs penetrate the cell membrane and localize in the nucleus without additional transfection vehicles.11 Numerous rationally designed PIPs have been developed as transcription repressors of therapeutically relevant genes, and they prevent transcription by inhibiting the binding of transcription factors to DNA.12 PIPs have also been supplemented with an epigenetically active molecule such as suberoylanilide-hydroxamic acid to generate synthetic genetic ON switches for cellfate-governing genes and noncoding RNAs.13 To date, PIPs and their conjugates have been extensively used to target nuclear DNA (nDNA) and to modulate endogenous expression of nuclear genes. Our notion is that PIPs can be developed as novel compounds to induce specific changes in the transcription status of the mitochondrial genome. However, the default accumulation of PIPs in the nuclei is a major concern. Furthermore, the highly dense, impermeable mitochondrial inner membrane containing saturated hydrophobic phospholipid molecules complicates the accurate delivery of PIPs into mitochondria. To overcome the delivery issue, we chose to employ a tunable peptide, termed mitochondria-penetrating peptide (MPP),14 composed of cyclohexylalanine (Cha) and Darginine (Arg), because MPP is known to redirect nucleilocalizing compounds such as doxorubicin and chlorambucil to mitochondria.15,16 The hydrophobicity of Cha reduces the energy barrier, and the positive charge of Arg provides the driving force for penetration, which depends on the membrane potential of the mitochondrial inner membrane.14 As a new paradigm to gain chemical control over mitochondrial transcription machinery, we chose to conjugate MPP with PIPs to
ABSTRACT: Synthetic ligands capable of recognizing the specific DNA sequences inside human mitochondria and modulating gene transcription are in increasing demand because of the surge in evidence linking mitochondrial genome and diseases. In the work described herein, we created a new type of mitochondria-specific synthetic ligand, termed MITO-PIPs, by conjugating a mitochondria-penetrating peptide with pyrrole-imidazole polyamides (PIPs). The designed MITO-PIPs showed specific localization inside mitochondria in HeLa cells and recognized the target DNA in a sequence-specific manner. Furthermore, MITO-PIPs that inhibit the binding of mitochondrial transcription factor A to the light-strand promoter (LSP) also triggered targeted transcriptional suppression. The tunability of PIPs’ properties suggests the potential of the MITO-PIPs as potent modulators of not only mitochondrial gene transcription but also its DNA mutations.
M
itochondria are essential organelles that play a pivotal role in several cellular functions, in addition to their most familiar roles in energy production and programmed cell death. In humans, impaired mitochondrial metabolism and translation are known to be associated with several complex diseases.1,2 The mitochondrial genome has its own ca. 16.6 kbp double-stranded, circular DNA (mtDNA). Each DNA strand is categorized on the basis of its guanine content as a heavy (H) strand or a light (L) strand. mtDNA encompasses 37 genes encoding 13 essential subunits of the oxidative phosphorylation system. Mitochondrial gene expression is controlled by a series of dynamic, intertwined processes where transcription occurs polycistronically from three transcription initiation sites in mtDNA: the L-strand promoter (LSP), the H-strand promoter 1 (HSP1), and the H-strand promoter 2 (HSP2).3 HSP initiates the transcription of the H-strand and produces two rRNAs, 12 mRNAs, and eight tRNAs. In contrast, LSP, located near HSP, produces only one mRNA and eight tRNAs. Contemporary assay tools and techniques including gain- and loss-of-function studies have already resolved the fundamental mechanism of mitochondrial transcription.4 The acquired data from the in vitro and cell-based studies validate the existence of three transcription factors: mtRNA polymerase (POLRMT), © 2017 American Chemical Society
Received: May 20, 2017 Published: June 14, 2017 8444
DOI: 10.1021/jacs.7b05230 J. Am. Chem. Soc. 2017, 139, 8444−8447
Communication
Journal of the American Chemical Society generate a new type of mitochondria-specific PIP, termed MITO-PIP (Figure 1).
Figure 1. (A) Chemical structure of a representative mitochondriaspecific PIP (MITO-PIP) complemented with a mitochondriapenetrating peptide. (B) Schematic illustration of transformed localization preference of MITO-PIPs.
Figure 2. (A) Chemical architecture of the designed MITO-PIPs and the control PIP lacking mitochondria-penetrating peptide. (B) Schematic illustration of the MITO-PIPs’ binding sites. Effect of PIP-LSP or MITO-PIP-LSP (C) and MITO-PIP-HSP1 (D) on the relative expression ratio ([ND6]/[MT-16S]) in HeLa cells after 24 h treatment. The primers are listed in Table S1.
The core structures of all MITO-PIPs (Figure 2A) were synthesized by using an automated solid-phase synthesizer. PIP without MPP was also synthesized to delineate the effect of MPP (PIP-LSP). Substitution of β-alanine instead of Nmethylpyrrole at the central position of each PIP is expected to increase the affinity and selectivity by reducing structural strain to fit the minor groove of DNA.17 All PIPs were used after purification by liquid chromatography (Figure S1). To evaluate the bioefficacy of MITO-PIP, we screened the three mitochondrial transcription factors as target candidates. TFAM attracted our interest because it serves as an additional control layer of mitochondrial transcription. TFAM has two highmobility group (HMG) box domains capable of intercalating into the minor groove at two different sites in LSP and HSP1,18 and the C-terminal tail is required to activate transcription machinery.19 TFAM-mediated activation of transcription from LSP is known to be associated with the U-turn structure imposed by TFAM in its responsive element-binding site, which, in turn, brings the C-terminal tail closer to the transcription start site.20 In the case of HSP1, the U-turn structure is not required because, unlike the binding pattern in the LSP site, TFAM binds to the HSP1 site in the reverse orientation to the C-terminal HMG box located near the transcription start site.18 MITO-PIPs were designed to target the known binding site of TFAM in LSP and HSP1 (Figure 2B).18 The successful inhibition of TFAM binding to LSP or HSP1 by MITO-PIP should result in a reduction in the level of expression of a downstream gene. Mitochondrially encoded NADH dehydrogenase 6 (ND6) located downstream of LSP plays a key role in mitochondrial metabolism, respiratory electron transport, and ATP synthesis. Accordingly, ND6 is associated with several
mitochondrial disorders, including Leber’s hereditary optic neuropathy and mitochondrial myopathy.21 Hence, the effect of MITO-PIPs on the endogenous expression of ND6 was calculated by using quantitative polymerase chain reaction (qPCR). HeLa cells were used in this study because their mitochondria have been extensively studied and well documented. Furthermore, they are suitable for imaging of mitochondria. Although mtDNA in HeLa cells was reported to have numerous haplotypes, we confirmed that there was no reported mutation on the binding site of TFAM in LSP or the HSP1 site.22 Based on the initial optimization experiments, HeLa cells treated with PIP-LSP and the MITO-PIPs targeting LSP and HSP1 were harvested after 24 h. To normalize gene expression and validate the specificity, we used MT-16S located downstream of HSP1. As shown in Figure 2C, MITO-PIP-LSP reduced the relative expression ratio ([ND6]/[MT-16S]) by about 60% at 5 μM and 90% at 10 μM. In contrast, the PIPLSP that lacked MPP did not show any notable change in the level of gene expression, indicating that the introduction of MPP is required for PIP to exhibit bioactivity in mitochondria. Interestingly, MITO-PIP-HSP1 also did not show any notable transcriptional change (Figure 2D). Hence, targeted suppression of ND6 suggests the sequence-specific bioactivity of MITO-PIP-LSP inside living cells. The discrepancy in bioactivity of MITO-PIPs could be attributed to the influence of the turn of PIPs in changing the preference of W/W base pairs (W = A or T).23 Therefore, the affinity of MITO-PIP-HSP1 is expected to be weaker than that 8445
DOI: 10.1021/jacs.7b05230 J. Am. Chem. Soc. 2017, 139, 8444−8447
Communication
Journal of the American Chemical Society Table 1. Shift of Tm Values by PIP-LSP, MITO-PIP-LSP, and MITO-PIP-HSP1a PIP-LSP ODN1/2 ODN3/4
MITO-PIP-LSP
MITO-PIP-HSP1
Tm/°C
Tm/°C
ΔTm/°C
Tm/°C
ΔTm/°C
Tm/°C
ΔTm/°C
47.7 (±0.6) 48.7 (±0.2)
74.1 (±0.9) 58.2 (±0.2)
26.5 9.4
71.7 (±1.2) 50.0 (±1.3)
24.0 1.3
49.6 (±0.5) 49.8 (±1.4)
2.0 1.1
Averages of Tm values are calculated from three melting temperature analyses, and each standard deviation is indicated in parentheses. ΔTm = Tm(compound−DNA complex) − Tm(DNA). a
Figure 3. Live cell imaging of HeLa cells. HeLa cells were treated with MITO-PIP-TAMRA at a concentration of 2 μM for 24 h. Nuclei and mitochondria were visualized by Hoechst 33342 and CellLight Mitochondria-GFP, BacMam 2.0, respectively. Scale bars represent 10 μm.
of MITO-PIP-LSP. To clarify this notion and to evaluate the binding affinity of MITO-PIPs, the thermal stability of DNA and DNA-MITO-PIP complex was analyzed along with the control PIP-LSP by thermal melting temperature (Tm) analysis. Two dsDNA samples were prepared with the following sequences (binding sites are underlined): 5′-GCGAACAGTCACCC (ODN1) and 5′-GGGTGACTGTTCGC (ODN2), and 5′-GCTCCGAACCACAG (ODN3) and 5′-CTGTGGTTCGGAGC (ODN4). The ODN1/ODN2 pair and ODN3/ODN4 pair have one binding site (underlined) of the MITO-PIPs targeting LSP and HSP1, respectively. The results of Tm analysis (Table 1) corroborate the high selectivity and binding affinity of MITO-PIP-LSP to ODN1/2, which was comparable to that of PIP-LSP. MITO-PIP-HSP1 did not cause a significant Tm shift with ODN3/4, thereby indicating its low affinity toward the HSP1 targeting sequence. Together, the data show that the binding pattern of MITO-PIPs confirms the distinct gene-suppressing ability of MITO-PIPs LSP and HSP1. We carried out imaging studies to confirm that the changes in gene expression caused by MITO-PIPs arise because of their precise localization into mitochondria. Based on the expected mitochondrial localization mechanism of MITO-PIPs, we performed imaging using live cells to keep the mitochondrial membrane potential. We labeled MITO-PIP-LSP with TAMRA to generate MITO-PIP-TAMRA (the chemical structure is shown in Figure S1) and investigated their intracellular distribution in HeLa cells. The HeLa cells were imaged by a confocal laser microscope after 24 h treatment with MITO-PIPTAMRA at a concentration of 2 μM (Figure 3). Mitochondria and nuclei were visualized by CellLight Mitochondria-GFP, BacMam 2.0 (Thermo Fisher Scientific), and Hoechst 33342, respectively. Consistent with the gene expression studies, MITO-PIP-TAMRA showed strong signals in the cytosol, and the merged image clearly showed it overlapped the signal of mitochondria-GFP, thereby confirming that the MITO-PIPTAMRA are efficiently localized in the mitochondria. It is
important to note here that the MITO-PIPs did not accumulate inside the nuclei. In summary, we have developed a new type of mitochondriaspecific DNA-based synthetic ligand, called MITO-PIPs, that can recognize a particular sequence of mtDNA. The designed LSP-specific MITO-PIP inhibited binding of TFAM to the LSP site in a promoter-specific manner and validated the conclusion that our synthetic ligand can distinguish the target sequence in LSP from that in HSP1. Live cell imaging studies confirmed that MITO-PIPs could precisely localize into the mitochondria. Notably, MITO-PIPs are the first DNA-based programmable small molecules capable of inducing targeted suppression of ND6, which is an essential factor of the respiratory chain complex in mitochondria. Previously, targeting peptide nucleic acid (PNA) oligomers conjugated with triphenylphosphonium cation have been synthesized to manipulate mtDNA replication.24 Whereas the designed PNA oligomers repressed mtDNA replication under in vitro conditions, bioactivity inside living cells was not achieved. Given that MITO-PIPs can trigger promoter-specific transcription suppression in HeLa cells, they could be developed further to target and alter the transcription status of the specific gene of interest. MITO-PIPs also have merit over PNA oligomers in that the double-strand structure of DNA could be maintained. Also, a fluorene-labeled polyamide derivative was reported to localize to mitochondria of human ovarian adenocarcinoma cells.25 However, the DNArecognition property of this derivative was negligible, as the structure encompassed only three pyrroles. Furthermore, this localization strategy could not be extended to the longer PIPs because the fluorene moiety in this derivative lacked positive charge, thereby hampering the driving force required for membrane penetration. In contrast, MITO-PIPs could be used to read the extended length of the target DNA sequence as the incorporation of arginine moiety renders the positive charge. Because of the automated synthesis of MITO-PIPs, it is easy to vary the target sequence of PIP and MPP. The HSP1-targeting 8446
DOI: 10.1021/jacs.7b05230 J. Am. Chem. Soc. 2017, 139, 8444−8447
Journal of the American Chemical Society
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MITO-PIP reported in this study did not induce gene expression changes. Considering the previous binding affinity studies,23 the PIP variants with distinct turn structures may not improve the bioactivity of MITO-PIP toward HSP1. Nonetheless, a combinatorial set of PIPs targeting different DNA sequences could facilitate the targeted modulation of HSP1. Our future work will focus on developing knowledge-based MITO-PIPs capable of altering the expression of HSP1governed genes. Likewise, the flexibility is a prime advantage of MITO-PIPs because preferred function(s) could be bestowed by the supplementation of other bioactive small molecules targeting other signaling factors. It is also possible to adjust the hydrophobicity and the number of positive charges to maximize and facilitate mitochondrial localization. Compared with nuclear DNA, mtDNA has a higher mutation rate because of the reactive oxygen species that are formed as byproducts of the oxidative phosphorylation. Although most mutations in mtDNA have a minimal effect on cellular function, recent evidence has revealed that some mutations can cause mitochondrial diseases.26 Furthermore, the mtDNA haplotype is related to the risk of other diseases such as diabetes.27 Whereas patients with Leber’s hereditary optic neuropathy have a T-to-C mutation in the ND6 gene, patients with Leigh syndrome have a G-to-A mutation. The mutated mtDNA is a prime target for disease therapy because a higher percentage of mutated mtDNA exceeding a threshold can cause cellular defects and mitochondrial diseases. MITO-PIPs could also read the sequence of a mutated region and distinguish mutated mtDNA from normal mtDNA. Therefore, MITO-PIPs can be developed further to generate a new functional compound that can exert bioactivity on either a normal or a mutated mtDNA in a defined manner. Our proof-of-concept study provides a fresh platform that opens new avenues for DNA-based functional ligands that are capable of altering the mitochondrial genome in a sequence-specific manner.
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REFERENCES
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b05230. Experimental procedures, characterization data for 1−4, primer list for qPCR experiments, and representative denaturation profiles in the Tm analyses, including Figures S1 and S2 and Table S1 (PDF)
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AUTHOR INFORMATION
Corresponding Authors
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[email protected] ORCID
Hiroshi Sugiyama: 0000-0001-8923-5946 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
We thank JCRB cell bank for providing HeLa cells (JCRB9004). This work was supported by JSPS [16H06356 to H.S., 16K12896 and Kyoto University SPIRITS grant to G.N.P.]. 8447
DOI: 10.1021/jacs.7b05230 J. Am. Chem. Soc. 2017, 139, 8444−8447
