Bioorthogonal Chemical Signature Enabling Amplified Visualization of Cellular Oxidative Thymines

Abstract

Cellular oxidative thymines, 5-hydroxymethyluracil (5hmU) and 5formyluracil (5fU), are found in the genomes of a diverse range of organisms, the distribution of which profoundly influence biological processes and living systems. However, the distribution of cellular oxidative thymines has not been explored because of lacking both specific bioorthogonal labeling and sensitivity methods for single-cell analysis. Herein, we report a bioorthogonal chemical signature enabling amplified visualization of cellular oxidative thymines in single cells. The synthesized ATP-γ-alkyne, an ATP analogue with bioorthogonal tag modified on γ-phosphate can be specifically linked to cellular 5hmU by chemoenzymatic labeling. DNA with 5-alkynephosphomethyluracil were then clicked with azide (N 3 )-modified 5hmU-primer. Identification of 5fU is based on selective reduction from 5fU to 5hmU, subsequent chemoenzymatic labeling of the newly generated 5hmU, and cross-linking with N 3 -modified 5fU-primer via click chemistry. Then, all of the 5hmU and 5fU sites are encoded with respective circularized barcodes. These barcodes are simultaneously amplified for multiplexed single-molecule imaging. The above two kinds of barcodes can be simultaneously amplified for differentiated visualization of 5hmU and 5fU in single cells. We find these two kinds of cellular oxidative thymines are spatially organized in a cell-type-dependent style with cell-to-cell heterogeneity. We also investigate their multilevel subcellular information and explore their dynamic changes during cell cycles. Further, using DNA sequencing instead of fluorescence imaging, our proposed bioorthogonal chemical signature holds great potential to offer the sequence information of these oxidative thymines in cells and may provide a reliable chemical biology approach for studying the whole-genome oxidative thymines profiles and insights into their functional role and dynamics in biology.

Lighting Up Nucleic Acid Modifications in Single Cells with DNA-Encoded Amplification

“…As a demonstration, we applied BEA-FISH to the detection of 5hmU and 5fU. 1 In mammals, 5hmU is mainly generated through two processing mechanisms: thymine hydroxylation and 5hmC deamination. Conventional imaging methods are disabled to detect single-cell 5hmU as as result of its low abundance (low ppm level).…”

Labeling and sequencing nucleic acid modifications using bio-orthogonal tools

“…Yongxi Zhao et al utilized bio-orthogonal reactions to image nucleic acid modifications in fixed cells. 114,115 One example is the simultaneous imaging of 5fC and 5hmC. 115 After labeling 5fC with AI, ligation with primer sequences linked to azide and DBCO was performed.…”

“…The imaging of 5hmU and 5fU in cells was carried out comparably. 114 The 5hmU site was modified with specific primers by modifying it with an alkynyl group using 5-HMUDK and ATP-γ-alkyne as described previously, and then by linking it to an azide-modified sequence via a bio-orthogonal reaction. After reducing the 5fU site with NaBH 4 to create a new 5hmU site, the modification and ligation steps were repeated, and a different primer sequence was connected to the 5fU site.…”

Sequencing 5-Formyluracil in Genomic DNA at Single-Base Resolution

“…7,19,20 5fU is of great significance in gene replication and expression, and it might be an important potential epigenetic mark. 21 Recently, a series of chemical methods using sequencing, 15 LC−MS/MS, 2,22 fluorescent labeling, 23−29 imaging 30 were applied to detect 5fU. Nevertheless, other potential biological functions of 5fU remained unclear.…”

“…Recently, a series of chemical methods using sequencing, LC–MS/MS, , fluorescent labeling, − and fluorescent imaging were applied to detect 5fU. Nevertheless, other potential biological functions of 5fU remained unclear.…”