RESUMO
Rare tautomeric forms of nucleobases can lead to Watson-Crick-like (WC-like) mispairs in DNA, but the process of proton transfer is fast and difficult to detect experimentally. NMR studies show evidence for the existence of short-time WC-like guanine-thymine (G-T) mispairs; however, the mechanism of proton transfer and the degree to which nuclear quantum effects play a role are unclear. We use a B-DNA helix exhibiting a wGT mispair as a model system to study tautomerization reactions. We perform ab initio (PBE0/6-31G*) quantum mechanical/molecular mechanical (QM/MM) simulations to examine the free energy surface for tautomerization. We demonstrate that while the ab initio QM/MM simulations are accurate, considerable sampling is required to achieve high precision in the free energy barriers. To address this problem, we develop a QM/MM machine learning potential correction (QM/MM-ΔMLP) that is able to improve the computational efficiency, greatly extend the accessible time scales of the simulations, and enable practical application of path integral molecular dynamics to examine nuclear quantum effects. We find that the inclusion of nuclear quantum effects has only a modest effect on the mechanistic pathway but leads to a considerable lowering of the free energy barrier for the GT*âG*T equilibrium. Our results enable a rationalization of observed experimental data and the prediction of populations of rare tautomeric forms of nucleobases and rates of their interconversion in B-DNA.
Assuntos
Pareamento de Bases , Guanina , Aprendizado de Máquina , Simulação de Dinâmica Molecular , Prótons , Teoria Quântica , Timina , Guanina/química , Timina/química , DNA/química , TermodinâmicaRESUMO
Many biotherapeutic formats leverage antibody light chain affinity chromatography to enable robust manufacturing processes and to streamline process development. These include multi-specific antibody and antibody fragment platforms which are often designed for specific capture purification methods that can provide efficient removal of commonly expressed product-related impurities. Recently, several accounts of product-related impurity separation by leveraging binding avidity during affinity chromatography have been described in the literature. However, a more comprehensive evaluation of avidity-based separations, particularly for light chain affinity media with specificity for constant regions of antibody light chains, is valuable for development of emerging multi-specific and fragment antibody formats. Results in this work demonstrate the capability of camelid antibody-based light chain affinity media to separate asymmetric bispecific antibody heterodimers from impurities possessing more than one light chain of the same class that the media binds to, including mispaired variants, aggregates, and fragment impurities. Largest resolution for respective mispaired species were provided by CaptureSelect KappaXP and LambdaXP chromatography media. The addition of elution modifiers provided increased impurity separation, with CaptureSelect KappaXP requiring up to 500 mM concentrations of elution modifiers to produce substantial improvements to resolution, and LambdaXP showing much higher sensitivity. Isocratic elution methods developed for lambda light chain affinity chromatography media provided near complete removal of mispaired variants, and substantial removal of aggregates and fragment impurities. Addition of just 20 mM of elution modifiers such as NaCl are shown to drive increased binding strength and separation of heterodimer species from impurities on CaptureSelect LambdaXP. These results provide scalable and transferable methods for product-related impurity control for various biotherapeutic modalities by lambda light chain affinity chromatography.
Assuntos
Anticorpos Biespecíficos , Anticorpos Biespecíficos/química , Cloreto de Sódio , Cromatografia de Afinidade/métodos , Fragmentos de ImunoglobulinasRESUMO
DNA mismatch repair (MMR) corrects non-Watson-Crick basepairs generated by replication errors, recombination intermediates, and some forms of chemical damage to DNA. In MutS and MutL homolog-dependent MMR, damaged bases do not identify the error-containing daughter strand that must be excised and resynthesized. In organisms like Escherichia coli that use methyl-directed MMR, transient undermethylation identifies the daughter strand. For other organisms, growing in vitro and in vivo evidence suggest that strand discrimination is mediated by DNA replication-associated daughter strand nicks that direct asymmetric loading of the replicative clamp (the ß-clamp in bacteria and the proliferating cell nuclear antigen, PCNA, in eukaryotes). Structural modeling suggests that replicative clamps mediate strand specificity either through the ability of MutL homologs to recognize the fixed orientation of the daughter strand relative to one face of the replicative clamps or through parental strand-specific diffusion of replicative clamps on DNA, which places the daughter strand in the MutL homolog endonuclease active site. Finally, identification of bacteria that appear to lack strand discrimination mediated by a replicative clamp and a pre-existing nick suggest that other strand discrimination mechanisms exist or that these organisms perform MMR by generating a double-stranded DNA break intermediate, which may be analogous to NucS-mediated MMR.
Assuntos
Reparo de Erro de Pareamento de DNA , DNA/metabolismo , Bactérias/genética , Bactérias/metabolismo , Replicação do DNA , Eucariotos/genética , Eucariotos/metabolismo , HumanosRESUMO
Energetically unfavorable Watson-Crick (WC)-like tautomeric forms of nucleobases are known to introduce spontaneous mutations, and contribute to replication, transcription, and translation errors. Recent NMR relaxation dispersion techniques were able to show that wobble (w) Gâ¢U mispair exists in equilibrium with the short-lived, low-population WC-like enolic tautomers. Presently, we have investigated the wGâ¢U â WC-like enolic reaction pathway using various theoretical methods: quantum mechanics (QM), molecular dynamics (MD), and combined quantum mechanics/molecular mechanics (QM/MM). The previous studies on QM gas phase calculations were inconsistent with experimental data. We have also explored the environmental effects on the reaction energies by adding explicit water. While the QM-profile clearly becomes endoergic in the presence of water, the QM/MM-profile remains consistently endoergic in the presence and absence of water. Hence, by including microsolvation and QM/MM calculations, the experimental data can be explained. For the Gâ¢Uenolâ Genolâ¢U pathway, the latter appears to be energetically more favorable throughout all computational models. This study can be considered as a benchmark of various computational models of wGâ¢U to WC-like tautomerization pathways with and without the environmental effects, and may contribute on further studies of other mispairs as well.
Assuntos
Guanina/metabolismo , RNA/genética , Uracila/metabolismo , Pareamento Incorreto de Bases/genética , Pareamento de Bases/genética , Simulação por Computador , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação Puntual/genética , Teoria QuânticaRESUMO
Site-specific cysteine engineering, along with other genetic mutations, is broadly implemented in bispecific antibodies (bsAb). Thus far, homodimer, half hole antibody, one-light chain mispaired and light chain swapped variants have been reported as chain-pairing variants for the asymmetric IgG-like bispecific antibodies. Here we report a novel mispair in which the CH3 engineered cysteine on the hole heavy chain (HC) of a knob-into-hole (KiH) bsAb is linked to the engineered cysteine in CL through a disulfide bond, forming a LHL species in a bsAb construct. Due to its impact on bioactivity, it is critical to implement an analytical strategy to monitor this CQA and mitigate risk for the future products. A set of orthogonal physicochemical assays that include hydrophobic interaction chromatography (HIC), capillary electrophoresis sodium dodecyl sulfate (CE-SDS), reverse phase liquid chromatography ultra-performance chromatography mass spectrometry (RP-UPLC MS) and disulfide bond mapping have been utilized to monitor and characterize this chain-pairing impurity for manufacturing process control and product release. Our data shows the LHL mispair in condition medium (CM) is approximately 1.3 - 1.9%. LambdaFabSelect affinity chromatography removes two major chain-pairing variants in CM - i.e. the hole-hole homodimer and hole half-antibody, while retaining the LHL species. Process improvement in Capto Q (anion exchange) and HS50 (cation exchange) chromatography steps removes LHL to as low as 0.2% in the final product. We have demonstrated an orthogonal analytical methodology that is capable of characterizing and monitoring bsAb mispairing, suitable for use in manufacturing process control and product release, and can be potentially implemented for similar bsAb constructs with engineered disulfide bonds.
Assuntos
Anticorpos Biespecíficos , Imunoglobulina G , Cromatografia , Cisteína , Espectrometria de MassasRESUMO
Antibody disulfide bond (DSB) reduction during manufacturing processes is a widely observed phenomenon attributed to host cell reductases present in harvest cell culture fluid. Enzyme-induced antibody reduction leads to product fragments and aggregates that increase the impurity burden on the purification process. The impact of reduction on bivalent bispecific antibodies (BisAbs), which are increasingly entering the clinic, has yet to be investigated. We focused on the reduction and reoxidation properties of a homologous library of bivalent BisAb formats that possess additional single-chain Fv (scFv) fragments with engineered DSBs. Despite all BisAbs having similar susceptibilities to enzymatic reduction, fragmentation pathways were dependent on the scFv-fusion site. Reduced molecules were allowed to reoxidize with and without low pH viral inactivation treatment. Both reoxidation studies demonstrated that multiple, complex BisAb species formed as a result of DSB mispairing. Furthermore, aggregate levels increased for all molecules when no low pH treatment was applied. Combined, our results show that complex DSB mispairing occurs during downstream processes while aggregate formation is dependent on sample treatment. These results are applicable to other novel monoclonal antibody-like formats containing engineered DSBs, thus highlighting the need to prevent reduction of novel protein therapeutics to avoid diminished product quality during manufacturing.
Assuntos
Anticorpos Biespecíficos , Dissulfetos , Oxirredutases/metabolismo , Proteínas Recombinantes , Animais , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/metabolismo , Reatores Biológicos , Células CHO , Cricetinae , Cricetulus , Dissulfetos/química , Dissulfetos/metabolismo , Contaminação de Medicamentos/prevenção & controle , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Oxirredução , Agregados Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/normas , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/metabolismoRESUMO
Minidumbbell (MDB) is a newly found non-B DNA structure formed by short single-strand sequences. Up to now, three MDBs have been reported to form at neutral pH by sequences containing two repeats of TTTA, CCTG and CTTG. Among them, the thermodynamically less stable TTTA and CCTG MDBs have been proposed to be the structural intermediates that cause TTTA and CCTG repeat expansions during DNA replication in Staphylococcus aureus pathogen and myotonic dystrophy type 2 patients, respectively. Although the CTTG MDB has a melting temperature of at least 13 °C higher than those of the other two, no CTTG repeat expansion has ever been reported in any genomes. In this study, we successfully determined the solution structure of the CTTG MDB and observed for the first time the formation of a reverse wobble T·T mispair with two symmetric hydrogen bonds. More importantly, we identified unprecedented hydrophobic interactions between the two methyl groups of this T·T mispair and the four 2'-methylene groups of their nearby loop-closing base pair residues. These stabilizations account for the substantial increase in the MDB thermodynamic stability which may govern the occurrence of repeat expansions.Communicated by Ramaswamy H. Sarma [Formula: see text].
Assuntos
DNA , Pareamento de Bases , Sequência de Bases , Humanos , Interações Hidrofóbicas e Hidrofílicas , Conformação de Ácido NucleicoRESUMO
A good Morpholino experiment starts with oligos that have been carefully designed to minimize off-target RNA binding. Performing a successful, reproducible, and well-controlled Morpholino experiment requires oligos that are single stranded and in solution at a known concentration. The outcome of treatment with the oligo needs to be checked for specificity, that is, that the observed outcome is due to interaction with the intended RNA and not an interaction with an unexpected RNA. In this chapter, I will discuss Morpholino use mostly in the context of embryonic microinjection experiments, though many techniques and warnings will be applicable to cell culture or adult animal experiments as well. Controls are critical to a good experiment, but good techniques in designing, preparing, storing, and using the oligos can improve the strength and specificity of the knockdown. Finally, it is important to know the solution concentration of the oligo to ensure that the results are reproducible.
Assuntos
Morfolinos/genética , Oligonucleotídeos Antissenso/genética , Animais , Pareamento de Bases , Técnicas de Silenciamento de Genes , Marcação de Genes , Humanos , Microinjeções , Morfolinos/administração & dosagem , Morfolinos/química , Morfolinos/normas , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/normas , Preservação Biológica/métodos , Controle de Qualidade , Estabilidade de RNARESUMO
The aim of this review is to describe the reactions which lead to generation of 5-hydroxymethyluracil, as well as the repair processes involved in its removal from DNA, and its level in various cells and urine. 5-hydroxymethyluracil may be formed during the course of the two processes: oxidation/hydroxylation of thymine with resultant formation of 5-hydroxymethyluracil paired with adenine (produced by reactive oxygen species), and reacting of reactive oxygen species with 5-methylcytosine forming 5-hydroxymethylcytosine, followed by its deamination to 5-hydroxymethyluracil mispaired with guanine. However, other, perhaps enzymatic, mechanism(s) may be involved in formation of 5-hydroxymethyluracil mispaired with guanine. Indeed, this mispair may be also formed as a result of deamination of 5-hydroxymethylcytosine, recently described "sixth" DNA base. It was demonstrated that 5-hydroxymethyluracil paired with adenine can be also generated by TET enzymes from thymine during mouse embryonic cell differentiation. Therefore, it is possible that 5-hydroxymethyluracil is epigenetic mark. The level of 5-hydroxymethyluracil in various somatic tissues is relatively stable and resembles that observed in lymphocytes, about 0.5/10(6) dN in human colon, colorectal cancer as well as various rat and porcine tissues. Experimental evidence suggests that SMUG1 and TDG are main enzymes involved in removal of 5-hydroxymethyluracil from DNA. 5-hydroxymethyluracil, in form of 5-hydroxymethyluridine, was also detected in rRNA, and together with SMUG1 may play a role in rRNA quality control. To summarize, 5-hydroxymethyluracil is with no doubt a product of both enzymatic and reactive oxygen species-induced reaction. This modification may probably serve as an epigenetic mark, providing additional layer of information encoded within the genome. However, the pool of 5-hydroxymethyluracil generated as a result of oxidative stress is also likely to disturb physiological epigenetic processes, and as such may be defined as a lesion. Altogether this suggests that 5-hydroxymethyluracil may be either a regulatory or erroneous compound.
Assuntos
Reparo do DNA/genética , DNA/genética , Pentoxil (Uracila)/análogos & derivados , 5-Metilcitosina/química , Animais , Bacteriófagos/genética , Humanos , Hidroxilação/fisiologia , Camundongos , Oxirredução , Pentoxil (Uracila)/química , Pentoxil (Uracila)/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Timina/química , Timina/metabolismoRESUMO
Transcriptional regulation is a tightly regulated, vital process. The transcription factor cyclic AMP-response element-binding protein 1 (CREB1) controls â¼25% of the mammalian transcriptome by binding the CREB1 binding site consensus sequence (CRE) sequence (TGACGTCA). DNA lesions within CRE modulate CREB1 binding negatively and positively. Because appropriate DNA lesions also interact with base excision repair proteins, we investigated whether CREB1 and repair glycosylases compete with each other. We incubated 39-mer CRE-containing double-stranded oligonucleotides with recombinant CREB1 alone or with UNG2 or OGG1, followed by EMSA. The CpG islet within CRE was modified to contain a G/U or 8-oxoG (°G)/C mispair. OGG1 and CREB1 reversibly competed for CRE containing an °G/C pair. Also, OGG1 blocked CREB1 from dimerizing by 69%, even when total CREB1 binding was reduced only by 20-30%. In contrast, bound CREB1 completely prevented access to G/U-containing CRE by UNG2 and, therefore, to base excision repair, whereas UNG2 exposure prevented CREB1 binding. CREB1 dimerization was unaffected by UNG2 when CREB1 bound to CRE, but was greatly reduced by prior UNG2 exposure. To explore physiological relevance, we microinjected zebrafish embryos with the same oligonucleotides, as a sink for endogenous CREB1. As predicted, microinjection with unmodified or lesion-containing CRE, but not scrambled CRE or scrambled CRE with a G/U mispair, resulted in increased embryo death. However, only the G/U mispair in native CRE resulted in substantial developmental abnormalities, thus confirming the danger of unrepaired G/U mispairs in promoters. In summary, CREB1 and DNA glycosylases compete for damaged CRE in vitro and in vivo, thus blocking DNA repair and resulting in transcriptional misregulation leading to abnormal development.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Dano ao DNA , DNA Glicosilases/metabolismo , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Sequência Consenso , Reparo do DNA , Humanos , Multimerização Proteica , Proteínas Recombinantes/metabolismo , Peixe-ZebraRESUMO
The intrinsic capability of the homo-purine DNA base mispairs to perform wobbleâWatson-Crick/Topal-Fresco tautomeric transitions via the sequential intrapair double proton transfer was discovered for the first time using QM (MP2/DFT) and QTAIM methodologies that are crucial for understanding the microstructural mechanisms of the spontaneous transversions.
Assuntos
Pareamento Incorreto de Bases , Pareamento de Bases , Prótons , Purinas/química , DNA/química , DNA/genética , Ligação de Hidrogênio , Modelos Moleculares , Estrutura Molecular , Conformação de Ácido Nucleico , Mutação Puntual , Teoria Quântica , Estereoisomerismo , Termodinâmica , Timina/químicaRESUMO
Cytosine methylation at CpG dinucleotides is a central component of epigenetic regulation in vertebrates, and the base excision repair (BER) pathway is important for maintaining both the genetic stability and the methylation status of CpG sites. This perspective focuses on two enzymes that are of particular importance for the genetic and epigenetic integrity of CpG sites, methyl binding domain 4 (MBD4) and thymine DNA glycosylase (TDG). We discuss their capacity for countering C to T mutations at CpG sites, by initiating base excision repair of G · T mismatches generated by deamination of 5-methylcytosine (5mC). We also consider their role in active DNA demethylation, including pathways that are initiated by oxidation and/or deamination of 5mC.
Assuntos
Ilhas de CpG , Reparo do DNA , DNA/metabolismo , Endodesoxirribonucleases/química , Epigênese Genética , Timina DNA Glicosilase/química , 5-Metilcitosina/química , 5-Metilcitosina/metabolismo , DNA/química , Metilação de DNA , Desaminação , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Humanos , Modelos Moleculares , Oxirredução , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Timina DNA Glicosilase/genética , Timina DNA Glicosilase/metabolismoRESUMO
We have shown for the first time, connecting QM methods with QTAIM analysis and using the methodology of the sweeps of the energetical, electron-topological and geometrical parameters, that the tautomerisation of the wobble guanine·thymine (wG·T) DNA base mispair into the wG(*)·T(*) base mispair induced by the double proton transfer (DPT), which undergoes a concerted asynchronous pathway, is not mutagenic. The wG·T â wG(*)·T(*) DPT tautomerisation does not result in the transition of the G base into its mutagenic tautomeric form G(*) able to mispair with the T base within the Watson-Crick base pairing scheme. This observation is explained by the so-called quantum protection of the wG·T DNA base mispair from its mutagenic tautomerisation - the dynamical non-stability of the tautomerised wG(*)·T(*) base mispair and significantly negative value of the Gibbs free energy of activation for the reverse reaction of the wG·T â wG(*)·T(*) DPT tautomerisation.
Assuntos
Pareamento de Bases , Guanina/química , Timina/química , Pareamento Incorreto de Bases , Simulação por Computador , DNA/química , Ligação de Hidrogênio , Modelos Moleculares , Teoria Quântica , Estereoisomerismo , TermodinâmicaRESUMO
Herein, we first address the question posed in the title by establishing the tautomerization trajectory via the double proton transfer of the adenine·guanine (A·G) DNA base mispair formed by the canonical tautomers of the A and G bases into the A*·G* DNA base mispair, involving mutagenic tautomers, with the use of the quantum-mechanical calculations and quantum theory of atoms in molecules (QTAIM). It was detected that the A·G â A*·G* tautomerization proceeds through the asynchronous concerted mechanism. It was revealed that the A·G base mispair is stabilized by the N6H···O6 (5.68) and N1H···N1 (6.51) hydrogen bonds (H-bonds) and the N2H···HC2 dihydrogen bond (DH-bond) (0.68 kcal·mol(-1) ), whereas the A*·G* base mispair-by the O6H···N6 (10.88), N1H···N1 (7.01) and C2H···N2 H-bonds (0.42 kcal·mol(-1) ). The N2H···HC2 DH-bond smoothly and without bifurcation transforms into the C2H···N2 H-bond at the IRC = -10.07 Bohr in the course of the A·G â A*·G* tautomerization. Using the sweeps of the energies of the intermolecular H-bonds, it was observed that the N6H···O6 H-bond is anticooperative to the two others-N1H···N1 and N2H···HC2 in the A·G base mispair, while the latters are significantly cooperative, mutually strengthening each other. In opposite, all three O6H···N6, N1H···N1, and C2H···N2 H-bonds are cooperative in the A*·G* base mispair. All in all, we established the dynamical instability of the Ð*·G* base mispair with a short lifetime (4.83·10(-14) s), enabling it not to be deemed feasible source of the A* and G* mutagenic tautomers of the DNA bases. The small lifetime of the Ð*·G* base mispair is predetermined by the negative value of the Gibbs free energy for the A*·G* â A·G transition. Moreover, all of the six low-frequency intermolecular vibrations cannot develop during this lifetime that additionally confirms the aforementioned results. Thus, the A*·G* base mispair cannot be considered as a source of the mutagenic tautomers of the DNA bases, as the A·G base mispair dissociates during DNA replication exceptionally into the A and G monomers in the canonical tautomeric form.