Gene expression is inherently dynamic. Conventional differential gene expression (DRE) analysis, due to technical limitations, struggles to capture transient changes in complex transcriptional regulation and unstable non-coding RNAs (such as enhancer RNAs). In this context, RNA sequencing provides an important tool for studying RNA dynamics by locating transcription start sites and quantifying nascent RNA.
However, compared with traditional DGE analysis, the study of nascent RNA faces more challenges, mainly due to its Short half-life and low abundance Characteristics. In recent years, a variety of analytical methods specifically for nascent RNA have emerged. These technologies reveal the degree of transcriptional activity in the promoter region, especially the residence time of PolⅡ in the proximal end of the promoter under the transcriptional activation state, which is a key link in the regulation of gene expression. In addition, nascent RNA not only directly affects the transcription process, but its sequence and structural characteristics also regulate dynamic behaviors such as transcription elongation, pause and stagnation, and participate in the binding process of chromatin modifying enzymes and enhancer RNA. At present, nascent RNA-seq technologies aimed at distinguishing newly synthesized RNA from other RNA are mainly divided into three categories: Transcriptional run-on-based methods, PolⅡ immunoprecipitation (IP)-based techniques, and metabolic labeling methods.
1. Run-on method
Enrichment of nascent RNA from total RNA by incorporation of nucleotide analogs during transcription and measurement of transient transcriptional activity (Figure a). Global run-on sequencing (GRO-seq) and precision nuclear run-on sequencing (PRO-seq) utilize 5-bromouridine 5′-triphosphate (BrU) or biotin-labeled nucleotides, respectively, to incorporate into nascent RNA, thereby mapping the location and activity of active RNA polymerases across the transcriptome. In the experiments, nuclei are isolated, endogenous nucleotides are washed away, and exogenously labeled nucleotides are then added to resume transcription. Enrichment of nascent transcripts through immunoprecipitation or affinity chromatography enables high-resolution detection of transcriptional activity.
Due to limitations in the amount of labeled nucleotide incorporation, GRO-seq has a resolution of only 10-50 bp, hindering precise localization of the transcription start site (TSS). In contrast, PRO-seq achieves single-base resolution by terminating transcription after biotin nucleotide incorporation. Although the run-on method is conceptually simple (it only requires enrichment of RNA molecules incorporating modified nucleotides), the presence of background non-nascent RNA increases the read depth required for sequencing.
The application of these technologies has revealed the widespread presence of divergent or bidirectional transcription in promoter regions and clarified the role of enhancer RNA in gene expression regulation. Furthermore, by combining specific enrichment of 5′-capped RNA (e.g., GRO-cap, PRO-cap, or START-seq), the sensitivity and specificity of transcription initiation detection have been further improved, while reducing background signal interference from post-transcriptional capped RNA. These improved methods can also capture RNAs that may be processed and removed during transcription, providing a more comprehensive perspective on transcriptional dynamics.
2. Pol II immunoprecipitation
Such as Native Elongating Transcription Sequencing (NET-seq) and its mammalian chromatin improved version mNET-seq, Studying transcriptional dynamics through antibody-specific capture of Pol II-associated RNA These methods utilize antibodies against the FLAG tag (directed to FLAG-tagged Pol II) or against the C-terminal domain of Pol II to enrich nascent RNA from chromatin complexes for mapping transcription start sites.
Although these techniques can directly correlate RNA polymerase activity with nascent RNA, they have the following limitations:
2.1 Background RNA interference:
Nonspecific binding of non-nascent RNA to Pol II and background mRNA can increase sequencing requirements and obscure analysis results.
2.2 Specificity issues:
NET-seq may enrich non-nascent RNAs (such as tRNA and small nucleolar RNAs) that strongly bind to Pol II, leading to data contamination.
2.3 Experimental complexity:
Although mNET-seq can reveal the regulatory mechanism of CTD modification and locate nascent RNA to TSS through a variety of CTD antibodies, it requires more cells, higher sequencing costs, and relies on complex experimental processes.
3. Metabolic labeling
The nucleotide analog 4-thiouridine (4 sU) is used to label nascent RNA, thereby enabling the detection of transcriptional dynamics (Figure c) However, methods with longer labeling times label the majority of transcripts, limiting sensitivity. Transient transcriptome sequencing (TT-seq) and thiol (SH)-linked alkylation RNA metabolic sequencing (SLAM-seq) address this issue by targeting the 3′ end of RNA (i.e., the newly transcribed region near the RNA polymerase), reducing background signals from 5′ RNA.
3.1 TT-seq:
Labeling time is limited to 5 minutes, and only the 3′ ends of new transcripts are labeled.
Adding an RNA fragmentation step before biotin affinity purification can enrich labeled RNA and improve detection sensitivity.
3.2 SLAM-seq：
Integrate 3' mRNA-seq library preparation (also applicable to miRNA libraries) to directly sequence tagged nascent RNAs rather than the entire transcript.
After RNA extraction, iodoacetamide is added to alkylate the 4sU residues, inducing reverse transcription-dependent T>C nucleotide conversion (manifested as a "mutation" in sequencing), thereby precisely locating the 4sU integration site.
However, the low integration rate results in only a few 4sU sites being converted to cytosine, limiting the detection sensitivity.
3.3 TUC-seq and TimeLapse-seq:
Also uses T>C mutation analysis, but does not enrich for the 3' end.
Used to study transcriptional responses and RNA half-life measurements after cell perturbations.
To overcome the above difficulties, we developed a Click Chemistry Innovative nascent transcript capture and library construction kit for the reaction. 5-Ethynyluracil (EU) Nascent RNA in living cells is labeled with biotin through a click chemistry reaction. The nascent RNA is then specifically captured using streptavidin magnetic beads, and a library is constructed. Combined with experimental timelines, this allows for quantitative analysis of newly generated RNA and the study of RNA transcription levels and degradation.
For product details, please click: Nascent Transcript Kit
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