Nucleic acid probes tagged with detectable reporting groups (mostly fluorophores and haptens) and/or functional modifications have been broadly used to allow the visualization, isolation, quantification of DNA and RNA molecules in order to track and understand their in-vitro and in-vivo activities, as well as their interactions with other biomolecules. The readout of these nuclei acid probes is generated either from a direct measurement of fluorescence signals in DNA or RNA, or from an indirect measurement of hapten (e.g., biotin and digoxigenin) labels using a secondary reporter (Scheme 1). The tags can be used for precise quantification of the amplification or hybridization events (qPCR and MERFISH, respectively), or as separation handles for isolation and enrichment purposes (RNA pulldown assay).
Scheme 1. Labeling strategies for nucleic acids. Top: Direct incorporation of fluorescent groups. Bottom: Two-step labeling approach. Incorporation of a functional group for subsequent labeling of a secondary reporter.
In general, there are two ways to introduce these tags into nucleic acids. Using solid-phase phosphoramidite chemistry (What are Phosphoramidites? | BroadPharm), the modifications can be incorporated into desired positions along an oligonucleotide sequence in a site-specific manner with high efficiency. This approach is dictated by the tag’s compatibility with phosphoramidite chemistry cycle and its stability under the final cleavage and deprotection conditions. The yield of full-length product also drops as the size of the oligonucleotide increases.
Alternatively, modifications can be introduced using chemically modified nucleoside triphosphate building blocks facilitated by engineered DNA/RNA polymerases either in a template-dependent or template-independent reaction (Table 1). One can generate the desired product in a short time. The mild aqueous condition is friendly to many sensitive modifications. This approach is especially suitable for generating long DNA/RNA sequences with high-density functional modifications for signal detection and sample processing. In this blog, we are focusing on the applications of enzymatic labeling of nuclei acids in basic research and molecular diagnostics.
Table 1. Available options for enzymatic nucleic acid labeling.
DNA or RNA molecules can be synthesized enzymatically using modified nucleoside triphosphate bearing a biotin (Biotin nucleotide | BroadPharm) or fluorescent dye (Fluorescent labeled nucleotide | BroadPharm) by primer extension or end-labeling. The nucleic acid sequences can also be labeled in a 2-step approach where a modified nucleotide substrate with a small functional handle was first incorporated into DNA followed by installation of a reporting group at the modified site using proper chemistry (for example, amino-11-ddUTP | BroadPharm was added to the oligo 3’-end using TdT followed by NHS-ester labeling of fluorescent dyes, Figure 1).
Figure 1. Two-step labeling of oligonucleotide using amino-modified ddUTP. RNA, 2023, 29, 1274-1287.
This approach is especially suitable for clickable chemistry partners (Figure 2) with diversified reporting groups in high-throughput screening. Additionally, in certain circumstances where the direct enzyme-mediated incorporation of dye-labeled dNTPs is less efficient due to the steric hindrance of bulky modifications, 2-step conjugation can improve the labeling yield significantly. BroadPharm carries modified nucleotides with clickable handles: azide nucleotides, DBCO nucleotides, tetrazine nucleotides, and TCO nucleotides | BroadPharm.
Figure 2. Clickable chemistry partners.
Many of the labeled nucleic acid sequences are involved in hybridization assays, which are powerful tools to understand how genes are expressed and regulated in different biological environments. For example, Spatial and temporal expression patterns of target genes in fixed tissue samples can be directly visualized using in-situ hybridization (ISH and/or FISH, Figure 3 and 4).
Figure 3. A simplified diagram depicting fluorescent in situ hybridization (FISH). Cells, 2022, 11, 3079.
Figure 4. Multiplex RNA visualization in cells using ViewRNA FISH Assays from Wikipedia
In microarray technique, hybridization between DNA (or cDNA) from a biological sample to a pre-designed complementary DNA probe arranged on a slide enables analysis of gene expression levels. Isolated nucleic acid samples are labeled by dye-conjugated nucleotides | BroadPharm via enzymatic incorporation (nick translation for DNA and reverse transcription for RNA, respectively). In order to compare the relative expression level between two samples, each labeled with a different dye (Figure 5, red for the experiment, green for the control), the resulting image is analyzed by calculating the ratio of the two dyes.
Figure 5. A typical DNA microarray co-hybridization (2 dye) experiment (from Bitesizebio.com).
In contrast to assays conducted on fixed cells, live cell RNA imaging provides insights to understand the dynamic behavior of cellular RNAs’ lifecyle by tracking their processing, spatiotemporal distribution and transportation. For example, fluorescently labeled oskar RNA was microinjected into Drosophila oocytes to study its short- and long-range transport (Curr. Biol., 1997, 7, 326). In more recent studies, fluorescent nucleobase analogues were incorporated enzymatically into cellular RNAs in order to achieve minimal perturbance. By replacing cytosine with its fluorescent analogue tCO, an mRNA transcript encoding H2B:GFP protein was constructed and transfected into human cells to visualize its delivery and translation in real time (Figure 6, JACS, 2021, 143, 5413).
Figure 6. Snap-shot images from a confocal time-lapse experiment to monitor the intracellular trafficking of 75% tCO-labeled mRNA (red) introduced, by chemical transfection, into cells with an overexpression of mRFP-Rab5 to label early endosomes (orange). Resulting expression of H2B-GFP protein in the nucleus is shown in green. White arrows indicate discrete mRNA–lipid complexes; scale bars: 10 μm.
In all aforementioned examples, the labels were used for visualization or quantification purposes. There are many other examples where special labels were incorporated in nucleic acid fragments to facilitate the isolation and enrichment of DNA/RNA sequences interacting with other biomolecules. In a classic biotinylated RNA pulldown assay (Scheme 2), RNA transcripts containing biotinylated nucleotides were prepared. The fragments bound to proteins were pulled down from cell lysate by streptavidin beads and subjected to further analysis to identify specific interacting partners.
Scheme 2. Workflow of biotinylated RNA pulldown assay.
Many RNA-protein interactions can be studied in similar manners. For example, by taking advantage of chemically modified nucleotides (Figure 7, ACS Chem. Bio. 2022, 17, 768) as capturing tags, specific interactions between nascent RNA and active RNA polymerase were mapped along the genome using next-generation sequencing to reveal the location and quantity of RNA production at specific sites. The method offered a powerful tool to analyze dynamic transcriptional activity with high mapping resolution genome-wide.
Figure 7. The NTP analogs designed to mark nascent RNA: a6ATP, N6-allyladenosine triphosphate; BrUTP, 5-bromouridine triphosphate; Biotin-NTP, biotin-modified NTP.
Chemically modified nucleoside triphosphate analogues have played an indispensable role in DNA sequencing technology advancement spanning four decades (Nature, 2017, 550, 345). The first-generation sequencing is also known as Sanger sequencing, in which chain-terminating fluorescent nucleotides (ddNTPs | BroadPharm) are used to create 3’ dye-labeled DNA pieces that differ in length by just one single base. The position and identity of the end base is resolved based on the size and its fluorophore of the DNA fragment. As the powerhouse of decoding genomic blueprint, Sanger sequencing dominated the genomic field for roughly 30 years.
Figure 8. Structures of the nucleotide reversible terminators with 3’-O blocking group.
The sophisticated next-generation sequencing (NGS) technology based on massively-parallel platforms (e.g. Illumina) almost completely superseded Sanger sequencing due to its high throughput, high multiplexicity and lower cost. Unlike the first-generation nucleotide terminator, NGS uses reversible terminators (such as 3’-O-azidomethyl dNTPs in Figure 8) that allow DNA extension to stop and restart one base at a time. The signal from a cleavable fluorescent label is erased after imaging (Scheme 3). In this way each base was identified along the primer extension (sequencing by synthesis, SBS).
Scheme 3. Sequencing by synthesis (SBS) cycle using 3’-O-blocked reversible terminators.
In the-third generation sequencing, signals were generated and detected at single molecule level where optical sensing (fluorescence/luminescence) is no longer exclusively used for base calling. Platforms based on polymerase extension, such as Helicos and PacBio are still using fluorophore tagged nucleotide (Figure 9A and B, respectively) for signal detection. PabBio’s single molecule real-time sequencing can generate high-quality long reads (10-25Kb) that are suitable for de novo gene assembly, full length transcriptomic studies, and metagenomic profiling. Recently Roche launched Axelios platform based on sequencing by expansion (SBX). The technology relies on a highly modified nucleotide with an acid-sensitive 5’-phosphoramidate linkage (Figure 9C). The sample DNA sequence is converted into a measurable surrogate polymer (Xpandomer) that was translocated through a nanopore for sequence decoding based on ion flow patterns. This platform allows ultra-fast turnaround time and flexible scalability to process time-sensitive, clinical samples.
Figure 9. Structures of representative nucleotides used in single-molecule sequencing.
Chemically modified nucleotides have found broad applications in modern biotechnology such as molecular diagnostics and precise medicine. Combining effort from engineering, automation and artificial intelligence, powerful tools have been developed to facilitate fundamental research discovery, which in turn help advance scientific frontiers of human knowledge and lead to innovations for disease prevention and treatment.
BroadPharm provides high purity, modified and/or functionalized nucleosides and nucleotides with a wide variety of options to facilitate your research and discovery. Custom synthesis services are also available upon request.