Universal Pretreatment Development for Low-input Proteomics Using Lauryl Maltose Neopentyl Glycol.

In recent years, there has been a growing demand for low-input proteomics, particularly in the context of single-cell proteomics (SCP). In this study, we have developed a lauryl maltose neopentyl glycol (LMNG)-assisted sample preparation (LASP) method. This method effectively reduces protein and peptide loss in samples by incorporating LMNG, a surfactant, into the digestion solution and subsequently removing the LMNG simply via reversed phase solid-phase extraction. The advantage of removing LMNG during sample preparation for general proteomic analysis is the prevention of mass spectrometry (MS) contamination. When we applied the LASP method to the low-input SP3 method and on-bead digestion in coimmunoprecipitation-MS, we observed a significant improvement in the recovery of the digested peptides. Furthermore, we have established a simple and easy sample preparation method for SCP based on the LASP method and identified a median of 1175 proteins from a single HEK239F cell using liquid chromatography (LC)-MS/MS with a throughput of 80 samples per day.

Synthesis and chemical transformations of glycol nucleic acid (GNA) nucleosides.

Xeno nucleic acids (XNA) are an increasingly important class of hypermodified nucleic acids with great potential in bioorganic chemistry and synthetic biology. Glycol nucleic acid (GNA) is constructed from a three-carbon 1,2-propanediol (propylene glycol) backbone attached to a nucleobase entity, representing the simplest known XNA. This review is intended to present GNA nucleosides from a synthetic chemistry perspective-a perspective that serves as a starting point for biological studies. Therefore this account focuses on synthetic methods for GNA nucleoside synthesis, as well as their postsynthetic chemical transformations. The properties and biological activity of GNA constituents are also highlighted. A literature survey shows four major approaches toward GNA nucleoside scaffold synthesis. These approaches pertain to glycidol ring-opening, Mitsunobu, SN2, and dihydroxylation reactions. The general arsenal of reactions used in GNA chemistry is versatile and encompasses the Sonogashira reaction, Michael addition, silyl-Hilbert-Johnson reaction, halogenation, alkylation, cyclization, Rh-catalyzed N-allylation, Sharpless catalytic dihydroxylation, and Yb(OTf)3-catalyzed etherification. Additionally, various phosphorylation reactions have enabled the synthesis of diverse types of GNA nucleotides, dinucleoside phosphates, phosphordiamidites, and oligos. Furthermore, recent advances in GNA chemistry have resulted in the synthesis of previously unknown redox-active (ferrocenyl) and luminescent (pyrenyl and phenanthrenyl) GNA nucleosides, which are also covered in this review.

Acyclic (S)-glycol nucleic acid (S-GNA) modification of siRNAs improves the safety of RNAi therapeutics while maintaining potency.

Glycol nucleic acid (GNA) is an acyclic nucleic acid analog connected via phosphodiester bonds. Crystal structures of RNA-GNA chimeric duplexes indicated that nucleotides of the right-handed (S)-GNA were better accommodated in the right-handed RNA duplex than were the left-handed (R)-isomers. GNA nucleotides adopt a rotated nucleobase orientation within all duplex contexts, pairing with complementary RNA in a reverse Watson-Crick mode, which explains the inabilities of GNA C and G to form strong base pairs with complementary nucleotides. Transposition of the hydrogen bond donor and acceptor pairs using novel (S)-GNA isocytidine and isoguanosine nucleotides resulted in stable base-pairing with the complementary G and C ribonucleotides, respectively. GNA nucleotide or dinucleotide incorporation into an oligonucleotide increased resistance against 3'-exonuclease-mediated degradation. Consistent with the structural observations, small interfering RNAs (siRNAs) modified with (S)-GNA had greater in vitro potencies than identical sequences containing (R)-GNA. (S)-GNA is well tolerated in the seed regions of antisense and sense strands of a GalNAc-conjugated siRNA in vitro. The siRNAs containing a GNA base pair in the seed region had in vivo potency when subcutaneously injected into mice. Importantly, seed pairing destabilization resulting from a single GNA nucleotide at position 7 of the antisense strand mitigated RNAi-mediated off-target effects in a rodent model. Two GNA-modified siRNAs have shown an improved safety profile in humans compared with their non-GNA-modified counterparts, and several additional siRNAs containing the GNA modification are currently in clinical development.

Tailored Tolane-Perfluorotolane Assembly as Supramolecular Base Pair Replacement in DNA.

Arene-fluoroarene interactions offer outstanding possibilities for engineering of supramolecular systems, including nucleic acids. Here, we implement the tolane-perfluorotolane interaction as base pair replacement in DNA. Tolane (THH) and perfluorotolane (TFF) moieties were connected to acyclic backbone units, comprising glycol nucleic acid (GNA) or butyl nucleic acid (BuNA) building blocks, that were incorporated via phosphoramidite chemistry at opposite positions in a DNA duplex. Thermodynamic analyses by UV thermal melting revealed a compelling stabilization by THH/TFF heteropairs only when connected to the BuNA backbone, but not with the shorter GNA linker. Detailed NMR studies confirmed the preference of the BuNA backbone for enhanced polar π-stacking. This work defines how orthogonal supramolecular interactions can be tailored by small constitutional changes in the DNA backbone, and it inspires future studies of arene-fluoroarene-programmed assembly of DNA.

Chemistry of glycol nucleic acid (GNA): Synthesis, photophysical characterization and insight into the biological activity of phenanthrenyl GNA constituents.

The knowledge pertaining to the chemistry and biological activity of glycol nucleic acid (GNA) components, like nucleosides and nucleotides, is still very limited. Herein we report on the preparation of the uracil nucleoside (1) and nucleotide ester GNA (2). The compounds are functionalised with a luminescent phenanthrenyl group. In DMSO, 1 and 2 are brightly fluorescent, with emission maxima at 390 nm, nanosecond decay times (0.6 and 0.75 ns, respectively), and quantum yields of ca. 0.2. In the solid phase, they show excimeric emission, with maxima at 495 nm (1) and 432 nm (2), and decay times of 3.7 ns (1) and 2.9 ns (2). The anticancer activity of the GNA components, as well as gemcitabine hydrochloride, used as a reference drug, were examined in vitro against human cancer HeLa and Ishikawa cells, as well as against normal L929 cells, using a battery of biochemical assays. Furthermore, biodistribution imaging studies were carried out in HeLa cells, with luminescence confocal microscopy, which showed that the compounds localized mainly in the lipophilic cellular compartments. Nucleoside (1) and nucleotide ester (2) features two different anticancer activity profiles. At 24 h of treatment, the nucleoside acts mainly as a toxin and induces necrosis in HeLa cells, whereas the nucleotide ester exhibits pro-apoptotic activity. At longer treatment times (72 h), the nucleoside and the reference, gemcitabine hydrochloride, featured almost identical signs of anticancer activity, such as S-phase cell cycle arrest, proliferation inhibition, and apoptosis induction. In view of this data, one can hypothesize that despite the structural differences, the newly obtained phenanthrenyl GNA nucleoside (1) and gemcitabine may share a common mechanism of anticancer activity in HeLa cancer cells. The GNA components were also examined as antiplasmodial agents against Plasmodium falciparum, in vitro. Nucleoside (1) was found to be more potent than nucleotide (2), displaying activity in the low micromolar range. Furthermore, both phenanthrene derivatives were found to display resistance indices at least 9-fold lower than chloroquine diphosphate (CQDP).

How do protein domains of low sequence complexity work?

This review covers research findings reported over the past decade concerning the ability of low complexity (LC) domains to self-associate in a manner leading to their phase separation from aqueous solution. We focus our message upon the reductionist use of two forms of phase separation as biochemical assays to study how LC domains might function in living cells. Cells and their varied compartments represent extreme examples of material condensates. Over the past half century, biochemists, structural biologists, and molecular biologists have resolved the mechanisms driving innumerable forms of macromolecular condensation. In contrast, we remain largely ignorant as to how 10%-20% of our proteins actually work to assist in cell organization. This enigmatic 10%-20% of the proteome corresponds to gibberish-like LC sequences. We contend that many of these LC sequences move in and out of a structurally ordered, self-associated state as a means of offering a combination of organizational specificity and dynamic pliability to living cells. Finally, we speculate that ancient proteins may have behaved similarly, helping to condense, organize, and protect RNA early during evolution.

Cancer-Mitochondria Dual-Targeting Glycol/Ferrocenium-Based Polydopamine Nanoparticles for Synergistic Photothermal and Photodynamic Therapy.

A cancer-mitochondria dual-targeting nanoparticle based on lactose and ferrocenium derivatives conjugated polydopamine (PDA@Lac/Fc/Hyp) was constructed, which exhibited cancer-targeting and mitochondria-targeting ability deriving from lactose and ferrocenium derivatives due to the specific carbohydrate-protein interaction and cationic species properties, respectively. Moreover, PDA@Lac/Fc/Hyp showed great biocompatibility and phototherapeutic efficiency. This work displays a good example of constructing cancer-mitochondria dual-targeting nanoparticle for synergistic phototherapy.

A Stereoselective Synthesis of Fused Carbocycles with a cis-1,2-Diol Moiety by Desymmetrization: SmI2-Mediated Pinacol Coupling of meso-Cyclic 1,3-Diones.

Samarium diiodide (SmI2)-mediated desymmetrization of a meso-cyclic 1,3-dione pinacol coupling is described. The reaction proceeds with high stereoselectivity to provide fused carbocyclic compounds with three contiguous stereogenic centers featuring an all-carbon quaternary center and a cis-1,2-diol moiety.

Tuning Exciton Coupling of Merocyanine Nucleoside Dimers by RNA, DNA and GNA Double Helix Conformations.

Exciton coupling between two or more chromophores in a specific environment is a key mechanism associated with color tuning and modulation of absorption energies. This concept is well exemplified by natural photosynthetic proteins, and can also be achieved in synthetic nucleic acid nanostructures. Here we report the coupling of barbituric acid merocyanine (BAM) nucleoside analogues and show that exciton coupling can be tuned by the double helix conformation. BAM is a nucleobase mimic that was incorporated in the phosphodiester backbone of RNA, DNA and GNA oligonucleotides. Duplexes with different backbone constitutions and geometries afforded different mutual dye arrangements, leading to distinct optical signatures due to competing modes of chromophore organization via electrostatic, dipolar, π-π-stacking and hydrogen-bonding interactions. The realized supramolecular motifs include hydrogen-bonded BAM-adenine base pairs and antiparallel as well as rotationally stacked BAM dimer aggregates with distinct absorption, CD and fluorescence properties.

Overcoming GNA/RNA base-pairing limitations using isonucleotides improves the pharmacodynamic activity of ESC+ GalNAc-siRNAs.

We recently reported that RNAi-mediated off-target effects are important drivers of the hepatotoxicity observed for a subset of GalNAc-siRNA conjugates in rodents, and that these findings could be mitigated by seed-pairing destabilization using a single GNA nucleotide placed within the seed region of the guide strand. Here, we report further investigation of the unique and poorly understood GNA/RNA cross-pairing behavior to better inform GNA-containing siRNA design. A reexamination of published GNA homoduplex crystal structures, along with a novel structure containing a single (S)-GNA-A residue in duplex RNA, indicated that GNA nucleotides universally adopt a rotated nucleobase orientation within all duplex contexts. Such an orientation strongly affects GNA-C and GNA-G but not GNA-A or GNA-T pairing in GNA/RNA heteroduplexes. Transposition of the hydrogen-bond donor/acceptor pairs using the novel (S)-GNA-isocytidine and -isoguanosine nucleotides could rescue productive base-pairing with the complementary G or C ribonucleotides, respectively. GalNAc-siRNAs containing these GNA isonucleotides showed an improved in vitro activity, a similar improvement in off-target profile, and maintained in vivo activity and guide strand liver levels more consistent with the parent siRNAs than those modified with isomeric GNA-C or -G, thereby expanding our toolbox for the design of siRNAs with minimized off-target activity.

Neopentyl Glycol as a Scaffold to Provide Radiohalogenated Theranostic Pairs of High In Vivo Stability.

The high in vivo stability of 2,2-dihydroxymethyl-3-[18F]fluoropropyl-2-nitroimidazole ([18F]DiFA) prompted us to evaluate neopentyl as a scaffold to prepare a radiotheranostic system with radioiodine and astatine. Three DiFA analogues with one, two, or without a hydroxyl group were synthesized. While all 125I-labeled compounds remained stable against nucleophilic substitution, only a 125I-labeled neopentyl glycol was stable against cytochrome P450 (CYP)-mediated metabolism and showed high stability against in vivo deiodination. 211At-labeled neopentyl glycol also remained stable against both nucleophilic substitution and CYP-mediated metabolism. 211At-labeled neopentyl glycol showed the biodistribution profiles similar to those of its radioiodinated counterpart in contrast to the 125I/211At-labeled benzoate pair. The urine analyses confirmed that 211At-labeled neopentyl glycol was excreted in the urine as a glucuronide conjugate with the absence of free [211At]At-. These findings indicate that neopentyl glycol would constitute a promising scaffold to prepare a radiotheranostic system with radioiodine and 211At.

Quantifying the phase separation property of chromatin-associated proteins under physiological conditions using an anti-1,6-hexanediol index.

BACKGROUND: Liquid-liquid phase separation (LLPS) is an important organizing principle for biomolecular condensation and chromosome compartmentalization. However, while many proteins have been reported to undergo LLPS, quantitative and global analysis of chromatin LLPS property remains absent. RESULTS: Here, by combining chromatin-associated protein pull-down, quantitative proteomics and 1,6-hexanediol (1,6-HD) treatment, we develop Hi-MS and define an anti-1,6-HD index of chromatin-associated proteins (AICAP) to quantify 1,6-HD sensitivity of chromatin-associated proteins under physiological conditions. Compared with known physicochemical properties involved in phase separation, we find that proteins with lower AICAP are associated with higher content of disordered regions, higher hydrophobic residue preference, higher mobility and higher predicted LLPS potential. We also construct BL-Hi-C libraries following 1,6-HD treatment to study the sensitivity of chromatin conformation to 1,6-HD treatment. We find that the active chromatin and high-order structures, as well as the proteins enriched in corresponding regions, are more sensitive to 1,6-HD treatment. CONCLUSIONS: Our work provides a global quantitative measurement of LLPS properties of chromatin-associated proteins and higher-order chromatin structure. Hi-MS and AICAP data provide an experimental tool and quantitative resources valuable for future studies of biomolecular condensates.

Time-dependent effect of 1,6-hexanediol on biomolecular condensates and 3D chromatin organization.

BACKGROUND: Biomolecular condensates have been implicated in multiple cellular processes. However, the global role played by condensates in 3D chromatin organization remains unclear. At present, 1,6-hexanediol (1,6-HD) is the only available tool to globally disrupt condensates, yet the conditions of 1,6-HD vary considerably between studies and may even trigger apoptosis. RESULTS: In this study, we first analyzed the effects of different concentrations and treatment durations of 1,6-HD and found that short-term exposure to 1.5% 1,6-HD dissolved biomolecular condensates whereas long-term exposure caused aberrant aggregation without affecting cell viability. Based on this condition, we drew a time-resolved map of 3D chromatin organization and found that short-term treatment with 1.5% 1,6-HD resulted in reduced long-range interactions, strengthened compartmentalization, homogenized A-A interactions, B-to-A compartment switch and TAD reorganization, whereas longer exposure had the opposite effects. Furthermore, the long-range interactions between condensate-component-enriched regions were markedly weakened following 1,6-HD treatment. CONCLUSIONS: In conclusion, our study finds a proper 1,6-HD condition and provides a resource for exploring the role of biomolecular condensates in 3D chromatin organization.

Single-Molecule Study of Thermomyces lanuginosus Lipase in a Detergency Application System Reveals Diffusion Pattern Remodeling by Surfactants and Calcium.

Lipases comprise one of the major enzyme classes in biotechnology with applications within, e.g., baking, brewing, biocatalysis, and the detergent industry. Understanding the mechanisms of lipase function and regulation is therefore important to facilitate the optimization of their function by protein engineering. Advances in single-molecule studies in model systems have provided deep mechanistic insights on lipase function, such as the existence of functional states, their dependence on regulatory cues, and their correlation to activity. However, it is unclear how these observations translate to enzyme behavior in applied settings. Here, single-molecule tracking of individual Thermomyces lanuginosus lipase (TLL) enzymes in a detergency application system allowed real-time direct observation of spatiotemporal localization, and thus diffusional behavior, of TLL enzymes on a lard substrate. Parallelized imaging of thousands of individual enzymes allowed us to observe directly the existence and quantify the abundance and interconversion kinetics between three diffusional states that we recently provided evidence to correlate with function. We observe redistribution of the enzyme's diffusional pattern at the lipid-water interface as well as variations in binding efficiency in response to surfactants and calcium, demonstrating that detergency effectors can drive the sampling of lipase functional states. Our single-molecule results combined with ensemble activity assays and enzyme surface binding efficiency readouts allowed us to deconvolute how application conditions can significantly alter protein functional dynamics and/or surface binding, both of which underpin enzyme performance. We anticipate that our results will inspire further efforts to decipher and integrate the dynamic nature of lipases, and other enzymes, in the design of new biotechnological solutions.

Caffeic Acid Phenethyl Ester-Incorporated Radio-Sensitive Nanoparticles of Phenylboronic Acid Pinacol Ester-Conjugated Hyaluronic Acid for Application in Radioprotection.

We synthesized phenylboronic acid pinacol ester (PBPE)-conjugated hyaluronic acid (HA) via thiobis(ethylamine) (TbEA) linkage (abbreviated as HAsPBPE conjugates) to fabricate the radiosensitive delivery of caffeic acid phenetyl ester (CAPE) and for application in radioprotection. PBPE was primarily conjugated with TbEA and then PBPE-TbEA conjugates were conjugated again with hyaluronic acid using carbodiimide chemistry. CAPE-incorporated nanoparticles of HAsPBPE were fabricated by the nanoprecipitation method and then the organic solvent was removed by dialysis. CAPE-incorporated HAsPBPE nanoparticles have a small particle size of about 80 or 100 nm and they have a spherical shape. When CAPE-incorporated HAsPBPE nanoparticles were irradiated, nanoparticles became swelled or disintegrated and their morphologies were changed. Furthermore, the CAPE release rate from HAsPBPE nanoparticles were increased according to the radiation dose, indicating that CAPE-incorporated HAsPBPE nanoparticles have radio-sensitivity. CAPE and CAPE-incorporated HAsPBPE nanoparticles appropriately prevented radiation-induced cell death and suppressed intracellular accumulation of reactive oxygen species (ROS). CAPE and CAPE-incorporated HAsPBPE nanoparticles efficiently improved survivability of mice from radiation-induced death and reduced apoptotic cell death. We suggest that HAsPBPE nanoparticles are promising candidates for the radio-sensitive delivery of CAPE.

Total Synthesis of Nortopsentin D via a Late-Stage Pinacol-like Rearrangement.

Nortopsentin D is part of a class of bis(indole) alkaloids known for their biological activity, including inhibitory activity in tumoral cells and antifungal activity. Herein we describe the first total synthesis of nortopsentin D, in which amidine and dione undergo a pivotal condensation and subsequent cyclization via a pinacol-like rearrangement. This synthesis represents a unique strategy for the formation of 5,5-disubstituted (4H)-imidazol-4-one containing natural products, many of which have yet to succumb to total synthesis.

Regiochemistry-Driven Organic Electrochemical Transistor Performance Enhancement in Ethylene Glycol-Functionalized Polythiophenes.

Novel p-type semiconducting polymers that can facilitate ion penetration, and operate in accumulation mode are much desired in bioelectronics. Glycol side chains have proven to be an efficient method to increase bulk electrochemical doping and optimize aqueous swelling. One early polymer which exemplifies these design approaches was p(g2T-TT), employing a bithiophene-co-thienothiophene backbone with glycol side chains in the 3,3' positions of the bithiophene repeat unit. In this paper, the analogous regioisomeric polymer, namely pgBTTT, was synthesized by relocating the glycol side chains position on the bithiophene unit of p(g2T-TT) from the 3,3' to the 4,4' positions and compared with the original p(g2T-TT). By changing the regio-positioning of the side chains, the planarizing effects of the S-O interactions were redistributed along the backbone, and the influence on the polymer's microstructure organization was investigated using grazing-incidence wide-angle X-ray scattering (GIWAXS) measurements. The newly designed pgBTTT exhibited lower backbone disorder, closer π-stacking, and higher scattering intensity in both the in-plane and out-of-plane GIWAXS measurements. The effect of the improved planarity of pgBTTT manifested as higher hole mobility (µ) of 3.44 ± 0.13 cm2 V-1 s-1. Scanning tunneling microscopy (STM) was in agreement with the GIWAXS measurements and demonstrated, for the first time, that glycol side chains can also facilitate intermolecular interdigitation analogous to that of pBTTT. Electrochemical quartz crystal microbalance with dissipation of energy (eQCM-D) measurements revealed that pgBTTT maintains a more rigid structure than p(g2T-TT) during doping, minimizing molecular packing disruption and maintaining higher hole mobility in operation mode.

1,6-Hexanediol, commonly used to dissolve liquid-liquid phase separated condensates, directly impairs kinase and phosphatase activities.

The concept of liquid-liquid phase separation (LLPS) has emerged as an intriguing mechanism for the organization of membraneless compartments in cells. The alcohol 1,6-hexanediol is widely used as a control to dissolve LLPS assemblies in phase separation studies in diverse fields. However, little is known about potential side effects of 1,6-hexanediol, which could compromise data interpretation and mislead the scientific debate. To examine this issue, we analyzed the effect of 1,6-hexanediol on the activities of various enzymes in vitro. Already at 1% volume concentration, 1,6-hexanediol strongly impaired kinases and phosphatases and partly blocked DNA polymerases, while it had no effect on DNase activity. At concentrations that are usually used to dissolve LLPS droplets (5-10%), both kinases and phosphatases were virtually inactive. Given the widespread function of protein phosphorylation in cells, our data argue for a careful review of 1,6-hexanediol in phase separation studies.

Effects of 18F-fluorinated neopentyl glycol side-chain on the biological characteristics of stilbene amyloid-ß PET ligands.

INTRODUCTION: The 2,2-dihydroxymethyl-1-[18F]fluoropropane group, also called 18F-labelled neopentyl glycol side-chain, is a novel 18F-labelling group for positron emission tomography (PET) imaging agents. The aim of using this group is to develop simple purification with solid-phase extraction without high-performance liquid chromatography. However, the effects of the neopentyl 18F-labelling group on the characteristics of brain imaging agents are unknown. Here, we added this side-chain to compounds with an aminostilbene structure to evaluate their effects on the biological properties of aminostilbene as an amyloid-ß (Aß) radioligand. METHODS: Biodistributions of four novel 18F-labelled stilbene compounds with different lengths of polyethylene glycol (PEG) linkers, called [18F]Cpd-0, -1, -2, and -4, (PEG = 0, 1, 2, and 4), and [18F]AV-1 in normal mice were evaluated. Metabolite analysis of [18F]Cpd-0 and -1 was performed with mouse plasma and brain. A competitive binding assay of [18F]AV-1 binding to Aß1-42 fibrils was performed to determine the binding properties of the compounds. RESULTS: [18F]Cpd-0, -1, and -2 demonstrated moderate initial brain uptake in mice (3.1-4.2% injected dose/g at 2 min post-injection) followed by fast clearance, and in vivo defluorination of these compounds was negligible. [18F]Cpd-4 exhibited low brain uptake and high bone uptake. Compared with [18F]Cpd-1, the percentage of [18F]Cpd-0 in mouse brain was high at 10 min post-injection. A competitive binding assay revealed partial interference effects by the neopentyl glycol side-chain on binding of stilbene compounds to Aß1-42 fibrils. CONCLUSIONS: Aminostilbene compounds with two or fewer PEG linkers containing an 18F-labelled neopentyl glycol side-chain demonstrated preferable pharmacokinetic properties as a brain imaging radioligand in normal mice. These side-chains can be used as an alternative labelling group for imaging agents targeting the brain.

Structural analysis of the chicken FANCM-MHF complex and its stability.

FANCM is involved in eukaryotic DNA-damage recognition and activates the Fanconi anemia (FA) pathway through complex formation. MHF is one of the FANCM-associating components and contains a histone-fold DNA-binding domain. Loss of the FANCM-MHF interaction compromises the activation of the FA pathway, resulting in chromosomal instability. Thus, formation of the FANCM-MHF complex is important for function, but its nature largely remains elusive. Here, the aim was to reveal the molecular and structural basis for the stability of the FANCM-MHF complex. A recombinant tripartite complex containing chicken FANCM (MHF interaction region), MHF1 and MHF2 was expressed and purified. The purified tripartite complex was crystallized under various conditions and three different crystals were obtained from similar crystallization conditions. Unexpectedly, structure determination revealed that one of the crystals contained the FANCM-MHF complex but that the other two contained the MHF complex without FANCM. How FANCM dissociates from MHF was further investigated and it was found that the presence of 2-methyl-2,4-pentanediol (MPD) and an oxidative environment may have promoted its release. However, under these conditions MHF retained its complexed form. FANCM-MHF interaction involves a mixture of hydrophobic/hydrophilic interactions, and chicken FANCM contains several nonconserved cysteines within this region which may lead to aggregation with other FANCM-MHF molecules. These results indicate an unexpected nature of the FANCM-MHF complex and the data can be used to improve the stability of the complex for biochemical and structural analyses.