DRUG-seq Technology: Workflow, Principles, and Applications

In the field of drug development, traditional technologies are plagued by high costs and long timeframes, leading to the notorious "$1 billion, 10-year" R&D dilemma for new drug development. The emergence of Digital RNA Perturbation Sequencing (DRUG-seq) technology has brought a new ray of hope to drug development.

This article systematically examines the definition, core principles, technical workflow, application scenarios, comparative analysis with conventional technologies, and recent advancements in DRUG-seq. By synthesizing these aspects, the study comprehensively demonstrates the technology's unique value and potential to revolutionize drug development.

What is DRUG-seq Technology?

In the long and intricate process of drug development, traditional methods for drug screening and mechanism-of-action studies are like groping in the fog, being inefficient and costly. The advent of DRUG-seq technology is like a ray of light piercing through the darkness, bringing new hope to this field.

Technology Definition

Digital RNA with perturbation of Genes sequencing, or DRUG-seq for short, is an innovative high-throughput drug screening technology. It skillfully combines RNA sequencing technology with microplate technology, breaking through the limitations of traditional approaches. On the small experimental platform of a microplate, hundreds of compounds can be tested simultaneously. Through advanced sequencing methods, it precisely captures the subtle changes these compounds cause in the cellular transcriptome, offering unprecedented efficiency and precision for drug development.

Core Principles

The core of DRUG-seq technology lies in its molecular barcodes and well-position labels. During the experiment, each cell sample is given a specific molecular barcode, acting as an exclusive "ID card" for the cell. At the same time, each well on the microplate has its unique label. This way, in subsequent data analysis, the origin of each sample and the compounds it has been exposed to can be tracked. With this clever design, it's possible to accurately analyze the transcriptome changes induced by hundreds of compounds in a single experiment, greatly enhancing the experiment's efficiency and the reliability of the data.

Technology Value

For a long time, new drug development has been facing the daunting challenge of the "$1 billion, 10-year" dilemma. The long development cycle and high costs make it difficult for many promising drugs to enter the market smoothly. The emergence of DRUG-seq technology provides a powerful weapon to crack this conundrum. It can speed up the compound screening process, allowing researchers to identify substances with potential therapeutic effects from a large number of compounds in a shorter time. Meanwhile, through in-depth analysis of transcriptome changes, it can more quickly reveal the mechanism of action (MoA) of compounds, providing a solid theoretical foundation for subsequent drug optimization and clinical trials. This not only reduces the cost of new drug development but also significantly shortens the research and development time, enabling new drugs to benefit patients more quickly.

DRUG-seq workflow (Ye et al., 2018)

An Analysis of the DRUG-seq Technology Workflow

Gaining an in-depth understanding of the DRUG-seq technology workflow helps us better grasp its key operational points and advantages.

Experimental Steps

• Step 1: Cell Culture and Drug Treatment

Cells are carefully seeded into 96-well or 384-well plates. These microplates act like tiny "laboratories," providing a stable growth environment for the cells. Then, different compounds are added to various wells to simulate the effects of drugs on the body. In this way, the impacts of multiple compounds on cells can be observed simultaneously, offering a wealth of data for subsequent analysis.

• Step 2: Lysis and Reverse Transcription

Cells are lysed using poly-dT primers with molecular barcodes and well-position labels, and the mRNA within them is captured. This process is like opening a "door" to the genetic information in the cells, allowing researchers to access the gene expression patterns of the cells under the influence of drugs. The poly-dT primers can specifically bind to the poly-A tails of mRNA, separating the mRNA from the complex cellular components and preparing it for subsequent reverse transcription and sequencing.

• Step 3: Library Construction and Sequencing

The captured mRNA is reverse-transcribed to generate cDNA, and a cDNA library is constructed. These libraries are then pooled together, and quantitative analysis of gene expression changes is carried out using advanced sequencing technologies. The sequencing results are like a detailed "gene report," clearly showing the expression status of each gene under the action of different compounds, providing researchers with clues to gain deep insights into the mechanism of drug action.

Technological Advantages

• Cost-effectiveness: Compared to traditional RNA-seq technology, DRUG-seq technology has significant cost advantages. Traditional RNA-seq technology usually requires separate sequencing of each sample, which is very expensive. However, DRUG-seq technology, through the high-throughput design of microplates, can process hundreds of samples simultaneously in a single experiment, reducing sequencing costs by 100 times. This enables researchers to conduct larger-scale experiments within a limited budget, improving the efficiency and reliability of their studies.
• Sensitivity: DRUG-seq technology also boasts extremely high sensitivity, capable of detecting subtle transcriptome changes caused by low-low-concentration drugs. In the early stages of drug development, many compounds may only exert their effects at low concentrations, and traditional detection methods often struggle to capture these minor changes. With its advanced technical principles and precise data analysis capabilities, DRUG-seq technology can accurately detect these weak signals, providing strong support for researchers in discovering potentially effective drugs.

Applications of DRUG-seq

The DRUG-seq technology has demonstrated extensive and significant application value in the fields of drug development and biotechnology. In drug development, it serves as a powerful tool for the discovery and optimization of new drugs. By screening a large number of compounds, it can quickly identify substances with potential therapeutic effects, thus shortening the drug development cycle.

For instance, Ye et al. took various compounds and the osteosarcoma cell line (U2OS) as research objects and employed the DRUG-seq technology to study the impact of drugs on the cellular transcriptome. DRUG-seq is a high-throughput, low-cost transcriptome analysis method. By detecting the transcriptome changes in cells after drug treatment, it can infer the mechanism of action and potential targets of drugs. The study found that DRUG-seq could group 433 compounds into functional clusters based on their mechanisms of action, such as compounds related to signal transduction, translation, epigenetics, and the cell cycle.

Moreover, DRUG-seq can infer the potential mechanisms of action of compounds with unknown targets and compare the effects of compound treatment and CRISPR gene knockout on the same target. DRUG-seq provides an efficient and low-cost transcriptome analysis tool for drug discovery, accelerating the drug development process.

DRUG-seq performance is on par with standard population RNA-seq (Ye et al., 2018)

Chen and their research team centered their study on Lianhua Qingwen Capsules. They utilized the DRUG-seq technology alongside RNA-seq and pharmacodynamic data to establish a multi-level network encompassing "herbal ingredients, in-vivo compounds, targets, and pathways." First, they detected 505 compounds in rats through UPLC-HRMS. Subsequently, they conducted a quantitative analysis of 46 compounds using HPLC- MS/MS. Based on this analysis, they selected 15 compounds with high exposure levels and significant activity potential as potential quality markers. These markers included well-known substances like ephedrine, amygdalin, and caffeic acid.

In an LPS-induced mouse pneumonia model, these selected markers demonstrated notable inhibitory effects. The research team predicted the mechanism of action through RNA-seq and further verified it using RT-qPCR. This study not only shed light on the overall mechanism of action of Lianhua Qingwen Capsules but also introduced a novel approach for enhancing its quality control. It sets a new benchmark for the quality control and mechanism research of traditional Chinese medicine compound preparations.

Evaluate compounds with potential immune or inflammatory regulatory effects in LHQW based on DRUG-seq data (Chen et al., 2025)

Li and their team focused on a variety of compounds and human-stem-cell-derived neurons for their research. They employed the DRUG-seq technology to investigate the biological activity of these compounds on the nervous system. DRUG-seq is a low-cost, high-throughput transcriptome sequencing method. It can directly lyse cells in 384-well plates and perform RNA sequencing.

In the study, the effects of 14 compounds with different mechanisms of action (MoA) on the U2OS cell line across an eight-point dose range were analyzed in detail. Additionally, compounds in a neuroscience drug discovery project targeting NMDA receptor agonists were also studied. The results showed that DRUG-seq had high reproducibility across different batches and plates. It could effectively classify compounds based on their mechanisms of action. In the research on NMDA receptor agonists, DRUG-seq not only detected the expected target-activation signals but also found independent off-target effects, demonstrating its potential in polypharmacology studies. Moreover, it could detect dose-dependent transcriptome changes in compounds, providing strong support for polypharmacology research.

DRUG-seq technology offers an unbiased biological activity readout method for neuroscience drug discovery. It enables rapid compound screening, mechanism of action elucidation, and the identification of potential off-target effects at the early discovery stage. This approach helps improve the efficiency of drug discovery, saving time and resources and provides new tools and ideas for drug development in the neuroscience field.

DRUG-seq detects on- and off-target effects for NR2A Drug Discovery Program(Li et al., 2022)

Comparison between DRUG-seq and Traditional Technologies

A comparison of DRUG-seq technology with traditional methods highlights its advantages.

Advantages of RNA-Seq

• Throughput: RNA-seq typically analyzes a single sample at a time. Each experiment only allows researchers to study gene expression under one compound or one set of conditions. In contrast, DRUG-seq, with its high-throughput design based on microplates, can screen 384 compounds in a single experiment. This significantly boosts experimental efficiency. It enables researchers to test more compounds in a shorter time, thus accelerating the drug development process.
• Cost: In terms of cost, the sequencing cost per sample for RNA-seq technology typically exceeds 1,000. This cost is prohibitively high for large-scale drug screening experiments. In contrast, the cost per well for DRUG-seq technology ranges from 50 to $100, significantly reducing the total experimental expenses. This reduction enables researchers to conduct larger-scale experiments within limited budgets, thereby enhancing the feasibility and reliability of the research.4.2 Advantages over PRISM and L1000

Although PRISM and L1000 technologies are also used in drug development, they have certain limitations. L1000 can only detect a subset of marker genes and fails to provide comprehensive transcriptome information. In contrast, DRUG-seq has the capability of full-transcriptome coverage. It can detect changes in the expression of all genes in cells, offering researchers more comprehensive and accurate gene expression data. This enables researchers to gain a deeper understanding of how drugs affect cellular gene expression and uncover the mechanisms of drug action.

Summary and Outlook

The DRUG-seq technology is reshaping the early-stage drug development process with its unique advantages. By bridging phenotypic screening and molecular mechanism research, it tightly links phenotypic changes with gene expression alterations during the drug screening process, offering researchers a more comprehensive and in-depth research perspective.

In the phenotypic screening phase, it enables the rapid identification of compounds with potential therapeutic effects. In the molecular mechanism research phase, it can delve into the mechanisms of action of these compounds, providing strong support for every aspect of drug development. This integrated research approach significantly enhances the efficiency and success rate of drug development, allowing new drugs to move from the lab to the clinic more quickly and benefit a large number of patients.

To better promote the application of DRUG-seq technology in drug development and the biotechnology field, researchers can access open-source platforms (such as GitHub, Novartis-related open-source platforms, etc.) to obtain DRUG-seq analysis tools. These platforms offer a wealth of code and resources, helping researchers quickly get started and conduct in-depth data analysis. By fully utilizing these open-source tools, researchers can accelerate the implementation of their studies, fully unleash the potential of DRUG-seq technology, and make greater contributions to the development of drug development and biotechnology.

In conclusion, as an innovative high-throughput transcriptome analysis technology, DRUG-seq shows great potential and broad prospects in the fields of drug development and biotechnology. With the continuous development and improvement of technology, it is believed that it will play an even more important role in future scientific research, bringing breakthroughs and hopes to the cause of human health.