Peripheral substitution effects on unnatural base pairs: A case of brominated TPT3 to enhance replication fidelity.

The high fidelity poses a central role in developing unnatural base pairs (UBPs), which means the high pairing capacity of unnatural bases with their partners, and low mispairing with all the natural bases. Different strategies have been used to develop higher-fidelity UBPs, including optimizing hydrophobic interaction forces between UBPs. Variant substituent groups are allowed to fine tune the hydrophobic forces of different UBPs' candidates. However, the modifications on the skeleton of TPT3 base are rare and the replication fidelity of TPT3-NaM remains hardly to improve so far. In this paper, we reasoned that modifying and/or expanding the aromatic surface by Bromo-substituents to slightly increase hydrophobicity of TPT3 might offer a way to increase the fidelity of this pair. Based on the hypothesis, we synthesized the bromine substituted TPT3, 2-bromo-TPT3 and 2, 4-dibromo-TPT3 as the new TPT3 analogs. While the enzyme reaction kinetic experiments showed that d2-bromo-TPT3-dNaM pair and d2, 4-dibromo-TPT3TP-dNaM pair had slightly less efficient incorporation and extension rates than that of dTPT3-dNaM pair, the assays did reveal that the mispairing of 2-bromo-TPT3 and 2, 4-dibromo-TPT3 with all the natural bases could dramatically decrease in contrast to TPT3. Their lower mispairing capacity promoted us to run polymerase chain amplification reactions, and a higher fidelity of d2-bromo-TPT3-dNaM pair could be obtained with 99.72 ± 0.01% of the in vitro replication fidelity than that of dTPT3-dNaM pair, 99.52 ± 0.09%. In addition, d2-bromo-TPT3-dNaM can also be effectively copied in E. coli cells, which showed the same replication fidelity as that of dTPT3-dNaM in the specific sequence, but a higher fidelity in the random sequence context.

Designing a Light-Induced Glycosylation Reaction for Poststage Synthesis of ADP-2″-Deoxyribosyl Derivatives.

Modifications on the hydroxyl groups of ADP-ribosyl units can provide valuable tools for investigating ADP-ribosylation-related molecular interactions, but the chemical syntheses of these compounds are usually difficult due to their inherent complex structures. In this study, we report a poststage synthetic protocol for accessing novel ADP-2″-deoxyribosyl derivatives through designing a light-induced biomimetic reaction, and SPR assays revealed effective binding of ADP-2″-deoxyribosyl peptides to MacroH2A1.1 with a high affinity (KD = 3.75 × 10-6 M).

Locating, tracing and sequencing multiple expanded genetic letters in complex DNA context via a bridge-base approach.

A panel of unnatural base pairs is developed to expand genetic alphabets. One or more unnatural base pairs (UBPs) can be inserted to enlarge the capacity, diversity, and functionality of canonical DNA, so monitoring the multiple-UBPs-containing DNA by simple and convenient approaches is essential. Herein, we report a bridge-base approach to repurpose the capability of determining TPT3-NaM UBPs. The success of this approach depends on the design of isoTAT that can simultaneously pair with NaM and G as a bridge base, as well as the discovering of the transformation of NaM to A in absence of its complementary base. TPT3-NaM can be transferred to C-G or A-T by simple PCR assays with high read-through ratios and low sequence-dependent properties, permitting for the first time to dually locate the multiple sites of TPT3-NaM pairs. Then we show the unprecedented capacity of this approach to trace accurate changes and retention ratios of multiple TPT3-NaM UPBs during in vivo replications. In addition, the method can also be applied to identify multiple-site DNA lesions, transferring TPT3-NaM makers to different natural bases. Taken together, our work presents the first general and convenient approach capable of locating, tracing, and sequencing site- and number-unlimited TPT3-NaM pairs.

Site-specific unnatural base excision via visible light.

Base excision (BE) is an important yet hard-to-control biological event. Unnatural base pairs are powerful tools to revolutionize biological studies in various areas. In this paper, we report a visible-light-induced method to construct site-specific unnatural BE and show the influence of its regulation on transcription and translation levels.

Amplification, Enrichment, and Sequencing of Mutagenic Methylated DNA Adduct through Specifically Pairing with Unnatural Nucleobases.

3-Methylthymine (m3T) is a long-known chemically stable but strongly mutagenic DNA base adduct; however, the sequencing method to determine m3T is lacking so far. Two of the main obstacles include the capacity of m3T to stall DNA elongation and its low abundance. To address the challenges, we report an unnatural base pairing strategy in this paper. A new m3T-TPT3 base pair was developed with a Vmax/Km value 82-fold higher than that of the m3T-A pair. The TPT3 nucleobase can be specifically incorporated opposite the m3T base, and the resulting m3T-TPT3 base pair can be effectively extended to give full-length products in the presence of commercial DNA polymerases. Importantly, the feature of the m3T-TPT3 pair enables a replacement PCR amplification to transfer m3T lesions at the exact positions into unnatural base pairs, which permits Sanger sequencings as well as biotin-streptavidin-based enrichments of m3T lesions. Taken together, this work offers a simple, convenient, and practical method for amplification, enrichment, and sequencing of m3T for the first time.

Mechanistic Insight into the Photoinduced Damage of an Unnatural Base Pair.

Chemical- and photostability of unnatural base pairs (UBPs) are important to maintain the genetic code integrity, and critical for developing healthy semisynthetic organisms. As reported, dTPT3 was less stable upon irradiation, and thus might act as a pervasive photosensitizer to induce oxidative damage within DNA, causing harm to living semi-synthetic organisms when exposed to UVA radiation. However, there was no knowledge about molecular-level understanding of this damage process. In this paper, we not only identified four photoproducts of dTPT3, including desulfur-dTPT3 (dTPT3H ), TPT3 sulphinate (TPT3SO2 ), TPT3 sulphonate (TPT3SO3 ) and TPT3-thioTPT3 (TPT3S TPT3), but also established a Type II photosensitized oxidation mechanism. In addition, the antioxidant (sodium ascorbate) was able to effectively inhibit the photoproducts formation of dTPT3 and dTPT3 in DNA, suggesting that a reductive environment might protect DNA bearing dTPT3 against UVA oxidation and ameliorate its adverse biological effects. The comprehensive understanding of TPT3' photochemical stability will give researchers helpful guidance to design more photostable UBPs and construct healthier semisynthetic organisms.

A chemical method for genome- and proteome-wide enrichment and O-GlcNAcylation profiling of chromatin-associated proteins.

O-Linked ß-N-acetylglucosamine (O-GlcNAc), a versatile posttranslational modification (PTM), is found on many chromatin-associated proteins (CAPs), such as transcription factors and their cofactors (TFCs). O-GlcNAc turnover influences the dynamic interactions of CAPs with chromatin and thereby regulates gene expression. Therefore, both global profiling of O-GlcNAc chromatin-associated proteins (OCAPs) and genome-wide mapping of their DNA binding sites are invaluable for understanding the functions of OCAPs and the regulatory machinery of O-GlcNAcylation on gene transcription. However, it is difficult to conduct genome- and proteome-wide OCAP studies using the widely adopted chromatin immunoprecipitation (ChIP) method due to the lack of highly O-GlcNAc-specific panantibodies. Therefore, we developed a chemical enrichment method (AFT-OCAP) for simultaneously profiling OCAPs and mapping their binding DNA via mass spectrometry (MS) analysis and DNA sequencing. In our method, we developed an alkynyl-functionalized trimethylpiperidine (AFT) reagent to perform highly efficient chemical derivatizations of azide-labeled OCAP-DNA complexes. The reversible affinity between the immobilized anti-trimethylpiperidine antibody resin and AFT reagent leads to specific enrichment and efficient elution of the OCAP-DNA complexes for both MS identification and sequencing. Deep coverage of OCAPs was achieved from HeLa cells, including 1951 O-GlcNAc peptides from 1136 O-GlcNAc chromatin-associated transcription factors and cofactors (TFCs) using HCD fragmentation and 669 O-GlcNAc sites using EThcD fragmentation. In addition, the distributions of O-GlcNAcylation across the genome and the dynamic interactions of OCAPs upon O-GlcNAc regulation were obtained.

Access to Photostability-Enhanced Unnatural Base Pairs via Local Structural Modifications.

Completing the storage and retrieval of increased genetic information in vivo and producing therapeutic proteins have been achieved by the unnatural base pair dNaM-dTPT3. Up to now, some biological and chemical approaches are implemented to improve the semi-synthetic organism (SSO). However, the photosensitivity of this pair, suggested as a potential threat to the healthy growth of cells, is still a problem to solve. Hence, we designed and synthesized a panel of TPT3 analogues with the basic structural skeletons of TPT3 but modified thiophene rings at variant sites to improve the photostability of unnatural base pairs. A comprehensive screening strategy, including photosensitivity tests, kinetic experiments, and replication in vitro by PCR and in vivo by amplification, was implemented. A new pair, dNaM-dTAT1, which had almost equally high efficiency and fidelity with the dNaM-dTPT3 pair itself both in vivo and in vitro, was proven to be more photostable and thermostable and less toxic to E. coli cells. The discovery of dNaM-dTAT1 represents our first progress for the optimization of this type of bases toward more photostable properties; our data also suggest that less photosensitive unnatural base pairs will be beneficial to build a healthier cellular replication system.

A triarylphosphine-trimethylpiperidine reagent for the one-step derivatization and enrichment of protein post-translational modifications and identification by mass spectrometry.

We report a new reagent that is capable of both chemical derivatization and selective enrichment of azide-labeled PTM peptides for sensitive identification by mass spectrometry (MS). Facile sample recovery, enhanced ionization and fragmentation in MS of the enriched PTM peptides are achieved, which leads to the identification of 3293 O-GlcNAc peptides and the location of 1706 sites in HeLa cells and efficiently expands the current mapping scale.

A sequential separation strategy for facile isolation and comprehensive analysis of human urine N-glycoproteome.

Urine is an attractive and non-invasive alternative source to tissue, blood or other biofluids for biomarker screening in clinical research. In normal human adult urine, 48% of the total urinary protein is in the sediment, 49% is soluble and the remaining 3% is contained in urinary extracellular vesicles (EVs). The soluble proteins and EV proteins in urine have attracted particular attention in recent years as cancer diagnostics. Furthermore, considering the important role of N-glycoproteins in practically all physiological processes, including regulating receptor-ligand binding, cell-cell interactions, inflammatory response and tumour progression, N-glycoproteome in human urine is an invaluable target for monitoring the physiological status and pathological changes of the kidney and urinary tract. Given the different origins of the soluble proteins and EV proteins in the urine, different N-glycoproteome patterns exist. Therefore, isolating the soluble N-glycoproteins and EV N-glycoproteins for separate analysis will provide a more specific and comprehensive view and provide a deeper understanding of human urinary N-glycoproteome. In this work, we developed a sequential separation method that isolates urinary soluble proteins and EV proteins via stepwise ultrafiltration based on their obvious size difference. A facile and reproducible protein isolation was achieved using this strategy. Subsequent N-glycoproteome enrichment and identification revealed distinct patterns in the two sub-proteomes of urine with more than 60% differential N-glycopeptides. A more comprehensive picture of the urinary N-glycoproteome with close to 1800 identified N-glycopeptides was obtained by this new analysis strategy, therefore making it advantageous for urinary biomarker screening. Graphical abstract A sequential separation method that isolates urinary soluble proteins and EV proteins via stepwise ultrafiltration was developed in this work. Subsequent N-glycopeptides enrichment and mass spectrometry analysis reveals distinct N-glycoproteome patterns in the two sub-proteomes of urine and a deep mapping of close to 1800 N-glycopeptides.