Mismatched Base Pairs Research Articles

Fluorogenic DNA-PAINT for faster, low-background super-resolution imaging.

DNA-based points accumulation for imaging in nanoscale topography (DNA-PAINT) is a powerful super-resolution microscopy method that can acquire high-fidelity images at nanometer resolution. It suffers, however, from high background and slow imaging speed, both of which can be attributed to the presence of unbound fluorophores in solution. Here we present two-color fluorogenic DNA-PAINT, which uses improved imager probe and docking strand designs to solve these problems. These self-quenching single-stranded DNA probes are conjugated with a fluorophore and quencher at the terminals, which permits an increase in fluorescence by up to 57-fold upon binding and unquenching. In addition, the engineering of base pair mismatches between the fluorogenic imager probes and docking strands allowed us to achieve both high fluorogenicity and the fast binding kinetics required for fast imaging. We demonstrate a 26-fold increase in imaging speed over regular DNA-PAINT and show that our new implementation enables three-dimensional super-resolution DNA-PAINT imaging without optical sectioning.

Exploring the Role of Msh2‐Msh6 in Processing Holliday Junction DNA

Msh2‐Msh6 is a eukaryotic ATP‐dependent mismatch repair (MMR) protein that plays a key role in maintaining the fidelity of the genetic code. The protein recognizes and initiates repair for single base pair mismatches and short insertion deletion loops (IDL). Msh2‐Msh6 functions via a molecular switch mechanism, whereby ATP hydrolysis and ADP/ATP exchange facilitates the binding and release of the protein from the mismatch. Previous research has revealed that the protein binds G:T mismatch DNA with the highest affinity. Interestingly, in‐solution binding assays have revealed that Msh2‐Msh6 binds Holliday Junction DNA, a key recombination intermediate, with 1:1 stoichiometry and a similarly high affinity. However, the functional importance and structural details of this interaction are unclear. Thus far, X‐ray crystallography and Molecular Dynamics (MD) simulations are limited to the protein’s interactions with mismatch DNA only. To address the lack of structural information, we modeled the Msh2‐Msh6‐HJ interaction using the human Msh2‐Msh6 crystal structure in which we have docked in junction DNA instead of the mismatched DNA. The junction DNA used in all experiments and simulations is J3, a non‐migrating junction with 17 base pair long arms. These MD simulations will help refine the structural model and assess significant interactions between the protein and DNA. Furthermore, ATPase assays and equilibrium binding assays are used to elucidate the protein’s ATP hydrolysis activity and binding affinity in the presence of G:T mismatch DNA, J3 junction DNA, and J3 junction DNA containing a G:T mismatch. Preliminary experiments show that relative to the apoprotein’s basal ATPase activity, ATP hydrolysis is weakly stimulated when Msh2‐Msh6 is bound to G:T mismatch and J3. Gel mobility shift assays (GMSA) reveal binding affinities in the range of 9.1±2.6 nM to 23.5±11.8 nM for G:T mismatch, J3 junction, and mismatched J3 junction. These data illustrate that Msh2‐Msh6 not only binds G:T mismatch and J3 with similarly high affinity, but also that introducing a mismatch in J3 does not modulate the protein’s affinity for the junction. In addition, the GMSA data suggest that Msh2‐Msh6‐HJ binding may take on multiple conformations, consistent with previous Förster resonance energy transfer (FRET) calculations that show Msh2‐Msh6 opening the junction after binding.

Encoded hydrogel microparticles with universal mismatch-incorporated DNA probes for highly specific multiplex detection of SNPs

Hydrogel microparticle-based nucleic acid assays are an attractive detection platform based on their multiplexing capabilities with high sensitivity and specificity. A particular area of interest is single-nucleotide polymorphism (SNP) sensing, where multiple SNPs should be identified in a highly reliable yet economical manner. However, hydrogel microparticles leveraging probe-target hybridization as a key mechanism are hampered by small duplex stability differences arising from single base-pair mismatch. We have developed encoded hydrogel microparticles with DNA probes tailored for multiplex SNP detection. Within the DNA probes, we adopt a widely used base analog (5-nitroindole) so that it substitutes one of the base sequences among DNA probes. The effects of the modification of the probes’ structure on SNP sensing has been tested from multiple perspectives, such as specificity, sensitivity, and available assay temperatures at a given ionic strength. We have validated that our hydrogel microparticles exhibit much higher specificity for a single base-pair mismatch with minimal reduction in sensitivity. Our particles can also detect multiple SNPs located in different target strands, which is a significant challenge for conventional particles.

Construction of Dual-Target Recognition-Based Specific MicroRNA Detection Method for Acute Pancreatitis Analysis.

MicroRNAs (miRNAs) play crucial roles in regulating various biological processes and are considered promising biomarkers for clinical diagnosis and therapy of acute pancreatitis. Herein, we present a duplex-specific nuclease (DSN enzyme) and DNAzyme-assisted fluorescent miRNA detections assay that can provide improved detection specificity due to a design of dual-target recognition and a comparable sensitivity. The dual-target recognitions are composed of (i) miRNA unfold hairpin structure toehold to form DNA-RNA duplex, among which the DNA section will be digested by DSN enzyme, releasing miRNA to participant in a next recycle. (ii) After DNAzyme-based nicking site formation in loop section of molecular beacon (MB), miRNA can bind with the loop section of MB and gradually unfold MB probe, generating fluorescence signals. With this general principle, distinct discrimination capability towards even one base pair mismatch of homogenous miRNA is obtained, showing a promising prospect in clinical diagnosis and therapy of acute pancreatitis.

The thumb subdomain of Pyrococcus furiosus DNA polymerase is responsible for deoxyuracil binding, hydrolysis and polymerization of nucleotides.

B-family DNA polymerases, which are found in eukaryotes, archaea, viruses, and some bacteria, participate in DNA replication and repair. Starting from the N-terminus of archaeal and bacterial B-family DNA polymerases, three domains include the N-terminal, exonuclease, and polymerase domains. The N-terminal domain of the archaeal B-family DNA polymerase has a conserved deoxyuracil-binding pocket for specially binding the deoxyuracil base on DNA. The exonuclease domain is responsible for removing the mismatched base pair. The polymerase domain is the core functional domain and takes a highly conserved structure composed of fingers, palm and thumb subdomains. Previous studies have demonstrated that the thumb subdomain mainly functions as a DNA-binding element and has coordination with the exonuclease domain and palm subdomain. To further elucidate the possible functions of the thumb subdomain of archaeal B-family DNA polymerases, the thumb subdomain of Pyrococcus furiosus DNA polymerase was mutated, and the effects on three activities were characterized. Our results demonstrate that the thumb subdomain participates in the three activities of archaeal B-family DNA polymerases as a common structural element. Both the N-terminal deoxyuracil-binding pocket and thumb subdomain are critical for deoxyuracil binding. Moreover, the thumb subdomain assists DNA polymerization and hydrolysis reactions, but it does not contribute to the fidelity of DNA polymerization.

Charge Transport in a Multiterminal DNA Tetrahedron: Interplay among Contact Position, Disorder, and Base-Pair Mismatch

As a secondary structure of DNA, DNA tetrahedra exhibit intriguing charge transport phenomena and provide a promising platform for wide applications like biosensors, as shown in recent electrochemical experiments. Here, we study charge transport in a multi-terminal DNA tetrahedron, finding that its charge transport properties strongly depend upon the interplay among contact position, on-site energy disorder, and base-pair mismatch. Our results indicate that the charge transport efficiency is nearly independent of contact position in the weak disorder regime, and is dramatically declined by the occurrence of a single base-pair mismatch between the source and the drain, in accordance with experimental results [J. Am. Chem. Soc. {\bf 134}, 13148 (2012); Chem. Sci. {\bf 9}, 979 (2018)]. By contrast, the charge transport efficiency could be enhanced monotonically by shifting the source toward the drain in the strong disorder regime, and be increased when the base-pair mismatch takes place exactly at the contact position. In particular, when the source moves successively from the top vertex to the drain, the charge transport through the tetrahedral DNA device can be separated into three regimes, ranging from disorder-induced linear decrement of charge transport to disorder-insensitive charge transport, and to disorder-enhanced charge transport. Finally, we predict that the DNA tetrahedron functions as a more efficient spin filter compared to double-stranded DNA and opposite spin polarization could be observed at different drains, which may be used to separate spin-unpolarized electrons into spin-up ones and spin-down ones. These results could be readily checked by electrochemical measurements and may help for designing novel DNA tetrahedron-based molecular nanodevices.

Cryo-EM structure of translesion DNA synthesis polymerase \u03b6 with a base pair mismatch

The B-family multi-subunit DNA polymerase ζ (Polζ) is important for translesion DNA synthesis (TLS) during replication, due to its ability to extend synthesis past nucleotides opposite DNA lesions and mismatched base pairs. We present a cryo-EM structure of Saccharomyces cerevisiae Polζ with an A:C mismatch at the primer terminus. The structure shows how the Polζ active site responds to the mismatched duplex DNA distortion, including the loosening of key protein-DNA interactions and a fingers domain in an “open” conformation, while the incoming dCTP is still able to bind for the extension reaction. The structure of the mismatched DNA-Polζ ternary complex reveals insights into mechanisms that either stall or favor continued DNA synthesis in eukaryotes.

Observing Mesoscopic Nucleic Acid Capacitance Effect and Mismatch Impact via Graphene Transistors.

This work reports a molecular-scale capacitance effect of the double helical nucleic acid duplex structure for the first time. By quantitatively conducting large sample measurements of the electrostatic field effect using a type of high-accuracy graphene transistor biosensor, an unusual charge-transport behavior is observed in which the end-immobilized nucleic acid duplexes can store a part of ionization electrons like molecular capacitors, other than electric conductors. To elucidate this discovery, a cascaded capacitive network model is proposed as a novel equivalent circuit of nucleic acid duplexes, expanding the point-charge approximation model, by which the partial charge-transport observation is reasonably attributed to an electron-redistribution behavior within the capacitive network. Furthermore, it is experimentally confirmed that base-pair mismatches hinder the charge transport in double helical duplexes, and lead to directly identifiable alterations in electrostatic field effects. The bioelectronic principle of mismatch impact is also self-consistently explained by the newly proposed capacitive network model. The mesoscopic nucleic acid capacitance effect may enable a new kind of label-free nucleic acid analysis tool based on electronic transistor devices. The in situ and real-time nucleic acid detections for virus biomarkers, somatic mutations, and genome editing off-target may thus be predictable.

PH dependence of C•A, G•A and A•A mismatches in the stem of precursor microRNA-31

MicroRNAs (miRNAs) are important regulators of post-transcriptional gene expression. Mature miRNAs are generated from longer transcripts (primary, pri- and precursor, pre-miRNAs) through a series of highly coordinated enzymatic processing steps. The sequence and structure of these pri- and pre-miRNAs play important roles in controlling their processing. Both pri- and pre-miRNAs adopt hairpin structures with imperfect base pairing in the helical stem. Here, we investigated the role of three base pair mismatches (A∙A, G∙A, and C∙A) present in pre-miRNA-31. Using a combination of NMR spectroscopy and thermal denaturation, we found that nucleotides within the three base pair mismatches displayed unique structural properties, including varying dynamics and sensitivity to solution pH. These studies deepen our understanding of how the physical and chemical properties of base pair mismatches influence RNA structural stability.

Simple construction of a two-component fluorescent sensor for turn-on detection of Hg2+ in human serum.

The simply constructed fluorescent sensor with inexpensive reagents and low toxicity has attracted increasing attention contributing to its practical application. However, the common construction methods usually required a few building blocks and complex procedures, which is inconvenient for their further application. Herein, a simply constructed fluorescent Hg2+ sensor has been developed based on the intrinsic fluorescence quenching power of G-quadruplex. Two components, AGRO 100 and AMT, were used to construct the sensor. AMT was selected as the fluorescent probe because of its distinct merits. The free AMT emits strongly. However, the fluorescence of AMT could be quenched by G-quadruplex DNA. Additionally, AMT is less toxic and inexpensive. AGRO 100 acts as both the quencher and the capture sequence because it consists of G-rich sequences and T-T mismatched base pairs. The fluorescence of AMT could be quenched by the formed G-quadruplex structure of AGRO 100 in the presence of K+. In the presence of Hg2+, G-quadruplex structure of AGRO 100 was switched to hairpin DNA structure because T-T mismatched base pairs in AGRO 100 could specifically recognize and capture Hg2+ with high affinity. Thus, AMT was released and the fluorescence of AMT was recovered. The developed sensing system was successfully applied to detect Hg2+ in human serum with good recovery and reproducibility.

Possibilities and challenges of small molecule organic compounds for the treatment of repeat diseases.

The instability of repeat sequences in the human genome results in the onset of many neurological diseases if the repeats expand above a certain threshold. The transcripts containing long repeats sequester RNA binding proteins. The mechanism of repeat instability involves metastable slip-out hairpin DNA structures. Synthetic organic chemists have focused on the development of small organic molecules targeting repeat DNA and RNA sequences to treat neurological diseases with repeat-binding molecules. Our laboratory has studied a series of small molecules binding to mismatched base pairs and found molecules capable of binding CAG repeat DNA, which causes Huntington’s disease upon expansion, CUG repeat RNA, a typical toxic RNA causing myotonic dystrophy type 1, and UGGAA repeat RNA causing spinocerebellar ataxia type 31. These molecules exhibited significant beneficial effects on disease models in vivo, suggesting the possibilities for small molecules as drugs for treating these neurological diseases.

Tandem Substitutions in Somatic Hypermutation.

Upon antigen recognition, activation-induced cytosine deaminase initiates affinity maturation of the B-cell receptor by somatic hypermutation (SHM) through error-prone DNA repair pathways. SHM typically creates single nucleotide substitutions, but tandem substitutions may also occur. We investigated incidence and sequence context of tandem substitutions by massive parallel sequencing of V(D)J repertoires in healthy human donors. Mutation patterns were congruent with SHM-derived single nucleotide mutations, delineating initiation of the tandem substitution by AID. Tandem substitutions comprised 5,7% of AID-induced mutations. The majority of tandem substitutions represents single nucleotide juxtalocations of directly adjacent sequences. These observations were confirmed in an independent cohort of healthy donors. We propose a model where tandem substitutions are predominantly generated by translesion synthesis across an apyramidinic site that is typically created by UNG. During replication, apyrimidinic sites transiently adapt an extruded configuration, causing skipping of the extruded base. Consequent strand decontraction leads to the juxtalocation, after which exonucleases repair the apyramidinic site and any directly adjacent mismatched base pairs. The mismatch repair pathway appears to account for the remainder of tandem substitutions. Tandem substitutions may enhance affinity maturation and expedite the adaptive immune response by overcoming amino acid codon degeneracies or mutating two adjacent amino acid residues simultaneously.

Coarse-grained molecular dynamics simulations of base-pair mismatch recognition protein MutS sliding along DNA

DNA mismatches are frequently generated by various intrinsic and extrinsic factors including DNA replication errors, oxygen species, ultraviolet, and ionizing radiation. These mismatches should be corrected by the mismatches repair (MMR) pathway to maintain genome integrity. In the Escherichia coli (E. coli) MMR pathway, MutS searches and recognizes a base-pair mismatch from millions of base-pairs. Once recognized, ADP bound to MutS is exchanged with ATP, which induces a conformational change in MutS. Previous single-molecule fluorescence microscopy studies have suggested that ADP-bound MutS temporarily slides along double-stranded DNA in a rotation-coupled manner to search a base-pair mismatch and so does ATP-bound MutS in a rotation-uncoupled manner. However, the detailed structural dynamics of the sliding remains unclear. In this study, we performed coarse-grained molecular dynamics simulations of the E. coli MutS bound on DNA in three different conformations: ADP-bound (MutSADP), ATP-bound open clamp (), and ATP-bound closed clamp () conformations. In the simulations, we observed conformation-dependent diffusion of MutS along DNA. MutSADP and diffused along DNA in a rotation-coupled manner with rare and frequent groove-crossing events, respectively. In the groove-crossing events, MutS overcame an edge of a groove and temporarily diffused in a rotation-uncoupled manner. It was also indicated that mismatch searches by is inefficient in terms of mismatch checking even though it diffuses along DNA and reaches unchecked regions more rapidly than MutSADP.

Fast Genotyping of K-Ras Codons 12 and 13 Based on Streptavidin Magnetic Microbeads Equipped with Biotin-Coupled Single Base-Pair Mismatch Pcr (Sm-Pcr)

Fast Genotyping of K-Ras Codons 12 and 13 Based on Streptavidin Magnetic Microbeads Equipped with Biotin-Coupled Single Base-Pair Mismatch Pcr (Sm-Pcr)

Investigation of unknown causes of uveal melanoma uncovers seven recurrent genetic mutations

Uveal melanoma (UM) is a rare subtype of melanoma but the most frequent primary cancer of the eye in adults. The goal of this study was to research the genetic causes of UM through a comprehensive frequency analysis of base-pair mismatches in patient genomes. It was hypothesized that chromosomes 15 and 16, due to their role in the expression of eye color, would exhibit UM-associated mutations. The U.S. National Institute of Health (NIH) provided genomic data from surgically resected eye enucleations and liver metastases from 32 UM patients, which we individually analyzed to identify nucleotide base-base mismatches by comparing DNA sequences from cancerous cells to reference DNA sequences. After identifying mismatches we performed a Pareto analysis of cross-patient data to identify chromosomes with recurrent and nonrecurrent genetic mutations across all samples. We then studied the mutated gene functions to evaluate causal links to cancer, such as for genes that encoded known tumor suppressor proteins. We discovered a total of 130 genetic mutations, including seven recurrent mutations, with most mutations occurring in chromosomes 3 and X. Recurrent mutations varied from 8.7% to 17.39% occurrence in the UM patient sample, with all mutations identified as missense. These findings suggest that UM is a recessive heterogeneous disease with selective homozygous mutations. Notably, this study has potential wider significance because the seven genes targeted by recurrent mutations are also involved in other cancers. As a result, immunotherapy is a highly promising treatment for uveal melanoma due to the disease’s heterogeneous nature.

Polyamine–Oligonucleotide Conjugates: 2′-OMe-Triazole-Linked 1,4,7,10-Tetraazacyclododecane and Intercalating Dyes and Their Effect on the Thermal Stability of DNA Duplexes

Oligonucleotides with the sequences 5′-GTG AUPA TGC, 5′-GCA TAUP CAC and 5′-GUPG ATA UPGC, where UP is 2′-O-propargyl uridine, were subjected to post-synthetic Cu(I)-catalyzed azide–alkyne cycloaddition to attach 1,4,7,10-tetraazacyclododecane (cyclen) and two well-known DNA intercalating dyes: thioxanthone and 1,8-naphthalimide. We propose a convenient cyclen protection–deprotection strategy that allows efficient separation of the resulting polyamine–oligonucleotide conjugates from the starting materials by RP-HPLC to obtain high-purity products. In this paper, we present hitherto unknown macrocyclic polyamine–oligonucleotide conjugates and their hybridization properties reflected in the thermal stability of thirty-two DNA duplexes containing combinations of labeled strands, their unmodified complementary strands, and strands with single base pair mismatches. Circular dichroism measurements showed that the B-conformation is retained for all dsDNAs consisting of unmodified and modified oligonucleotides. An additive and destabilizing effect of cyclen moieties attached to dsDNAs was observed. Tm measurements indicate that placing the hydrophobic dye opposite to the cyclen moiety can reduce its destabilizing effect and increase the thermal stability of the duplex. Interestingly, the cyclen-modified U showed significant selectivity for TT mismatch, which resulted in stabilization of the duplex. We conclude the paper with a brief review and discussion in which we compare our results with several examples of oligonucleotides labeled with polyamines at internal strand positions known in the literature.

RNA-DNA Hybrid Nanoshape Synthesis by Facile Module Exchange.

The preparation of nucleic acid nanostructures has relied predominantly on procedures of additive fabrication in which complex architectures are assembled by concerted self-assembly and sequential addition of building blocks. We had previously established RNA-DNA hybrid nanoshapes with modular architectures that enable multistep synthetic approaches inspired by organic molecular synthesis where additive and transformative steps are used to prepare complex molecular architectures. We report the establishment of module replacement and strand exchange as synthetic transformations in nucleic acid hybrid nanoshapes, which are enabled by minimally destabilizing sequence elements such as a single unpaired overhang nucleotide or a mismatch base pair. Module exchange facilitated by thermodynamic lability triggers adds a powerful transformative approach to the repertoire of additive and transformative synthetic methods for the preparation of complex composite materials.

Bending of Canonical and G/T Mismatched DNAs.

Mismatched base pairs alter the flexibility and intrinsic curvature of DNA. The role of such DNA features is not fully understood in the mismatch repair pathway. MutS/DNA complexes exhibit DNA bending, PHE intercalation, and changes of base-pair parameters near the mismatch. Recently, we have shown that base-pair opening in the absence of MutS can discriminate mismatches from canonical base pairs better than DNA bending. However, DNA bending in the absence of MutS was found to be rather challenging to describe correctly. Here, we present a computational study on the DNA bending of canonical and G/T mismatched DNAs. Five types of geometric parameters covering template-based bending toward the experimental DNA structure, global, and local geometry parameters were employed in biased molecular dynamics in the absence of MutS. None of these parameters showed higher discrimination than the base-pair opening. Only roll could induce a sharply localized bending of DNA as observed in the experimental MutS/DNA structure. Further, we demonstrated that the intercalation of benzene mimicking PHE decreases the energetic cost of DNA bending without any effect on mismatch discrimination.

Tandem Substitutions in Somatic Hypermutation

Upon antigen recognition, activation-induced cytosine deaminase initiates affinity maturation of the B-cell receptor by somatic hypermutation (SHM) through error-prone DNA repair pathways. SHM typically creates single nucleotide substitutions, but tandem substitutions may also occur. While tandem substitutions have been described in mice and other species, the incidence of this phenomenon and its underlying mechanism in humans is currently unknown.We investigated incidence and sequence context of tandem substitutions by massive parallel sequencing of V(D)J repertoires in healthy human donors generated by unbiased ARTISAN PCR. Selection of unique, clonally unrelated, antigen-experienced sequences carrying up to 5% mutations yielded 13.532 VDJ, 7.952 VJ-kappa and 7.598 VJ-lambda. Comparison to the closest germline allele allowed for identification of a total of 122.878 single nucleotide substitutions (SNS), 10.735 tandem dinucleotide substitutions (TDNS) and 2.615 longer contiguous substitutions. After correcting for expected clusters of adjacent SNS, tandem substitutions comprised 5,7% of all AID-induced mutations. The mutation of more than one nucleotide in a single event, was shown to overcome amino acid codon redundancy and may therefore enhance the adaptive immune response. Clustering of such mutations around AID hotspots and their overall distribution indicates that tandem substitutions are an integral part of the SHM spectrum.In the majority of tandem substitutions, the mutated sequence may be identified in the directly adjacent reference sequence context. Tandem substitutions in humans therefore represent single nucleotide juxtalocations. Such juxtalocations appear to be favored in polydipyramidine stretches. These observations could be confirmed in patients with MSH2/6 deficiency, but were absent in a VDJ library from an UNG-deficient patient, indicating a strict dependence on abasic sites as an instigating mechanism.Together, these findings delineate a model where tandem substitutions are predominantly generated by translesion synthesis across an apyramidinic site that is typically created by UNG. During replication, apyrimidinic sites transiently adapt an extruded configuration, causing skipping of the extruded base. Consequent strand decontraction leads to the juxtalocation, after which exonucleases repair the apyramidinic site and any directly adjacent mismatched base pairs. The mismatch repair pathway appears to account for the remainder of tandem substitutions.Our study shows that a significant portion of mutations acquired during SHM are caused by tandem substitutions, and that this mechanism may enhance affinity maturation and expedite the adaptive immune response by overcoming amino acid codon degeneracies or mutating two adjacent amino acid residues simultaneously.Figure legend. Corrected incidence of tandem dinucleotide substitutions in healthy donors. (A) Dinucleotide substitutions from unique IGHV, IGKV and IGLV sequences and corrected after in silico predictions of dinucleotide substitutions that did not occur in tandem. Burgundy cells represent sequence inversions, light and dark purple cells represent juxtalocations of the 5‘ and 3‘ base in the pair (as seen from the non-transcribed strand), respectively. For unshaded cells, juxtalocation could not be assessed due to one or more nucleotides in the reference sequence matching the mutated sequence. (B) Relative contribution of sequence inversions and juxtalocations. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Primary Complete Mitochondrial Genome of the Lappet Moth Brahmophthalma hearseyi (Lepidoptera: Brahmaeidae) and Related Phylogenetic Analysis.

Simple SummaryIn this paper, the complete mitochondrial genome (mitogenome) of B. hearseyi was sequenced using long-PCR and primer-walking methods. The results indicated that the mitogenome is a typical circular molecule that is composed of 15,442 bp. Phylogenetic analysis showed that B. hearseyi is clustered into Brahmaeidae, and the phylogenetic relationships are (Brahmaeidae + Lasiocampidae) + (Bombycidae + (Sphingidae + Saturniidae)). This study provides the first mitogenomic resources for the Brahmaeidae.Background: Brahmophthalma hearseyi (Lepidoptera: Brahmaeidae) is widely distributed across China. Its larvae damage the leaves of many plants such as those belonging to the Oleaceae family, causing significant economic losses and seriously affecting the survival and reproduction of Cervus nippon; however, genetic data for this species are scarce. Methods: The complete mitochondrial genome (mitogenome) of B. hearseyi was sequenced using long-PCR and primer-walking methods. Phylogenetic analysis that was based on 13 PCGs and two rRNAs was carried out using the neighbor-joining and Bayesian interference methods. Results: The mitogenome is a typical circular molecule that is made up of 15,442 bp, which includes 13 protein-coding genes (PCGs), 2 ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and an A + T-rich region (456 bp). All of the PCGs, except for COX1 and COX2, start with ATN codons. COX2 and ND5 use the incomplete termination codon T, and 11 other PCGs use the typical stop codon TAA. All tRNA genes, except for trnS1 and trnS2, display a typical cloverleaf structure; trnS1 lacks the “DHU” arm, whereas trnS2 exhibits two mismatched base pairs in the anticodon stem. Phylogenetic analysis showed that B. hearseyi is clustered into Brahmaeidae, and the phylogenetic relationships are (Brahmaeidae + Lasiocampidae) + (Bombycidae + (Sphingidae + Saturniidae)). Conclusions: This study provides the first mitogenomic resources for the Brahmaeidae.