In the field of epigenomics, accurate detection of N6-methyladenosine (m6A) modification is always the core proposition to analyze its biological function. Based on the principle of antibody-independent enzymatic demethylation, GLORI 1.0 established the gold standard status of m6A detection by virtue of single-base resolution and quantitative ability, and provided a breakthrough tool for revealing the functional specificity of modification sites. However, its high RNA input demand (microgram level), complex operation process, and potential incomplete transformation risk limit its application in rare samples (such as clinical biopsy tissues and rare cell populations) and the standardization and popularization of the technology.

The birth of GLORI 2.0 marks the great-leap-forward evolution of this gold standard. Through the development of a new high-activity recombinant ALKBH5 enzyme, the systematic simplification of the experimental process, and the precise optimization of reaction conditions, this technology can reduce the RNA input to the nanogram level, significantly shorten the operation time, and achieve near-perfect transformation efficiency while retaining the advantages of single-base resolution and quantification.

The technical transition from GLORI 1.0 to 2.0 not only solved the practical bottleneck of the original method but also expanded the boundary of epigenomics research, making it move from batch sample analysis to high-precision detection of trace, single-cell, and clinical samples, which provided stronger methodological support for basic research and transformation application in this field.

The article describes the evolution of GLORI from 1.0 to 2.0, highlighting 1.0's role as a gold standard, its limitations, and 2.0's innovations in reducing RNA input, simplifying workflow, and improving efficiency.

Establishing the Benchmark with GLORI 1.0

As a milestone technology in the field of epigenetics, the pioneering principle of GLORI 1.0 completely changed the detection paradigm of m6A modification. The technology abandons the traditional antibody-dependent detection mode and innovatively adopts an enzymatic demethylation reaction, that is, the human ALKBH5 demethylase is used to specifically catalyze the transformation of m6A into adenosine (A), and by comparing the high-throughput sequencing data of the enzyme-treated group and the untreated group, the single-base resolution m6A locus location is realized.

This breakthrough in principle has brought multiple advantages:

• On the one hand, getting rid of antibody dependence avoids false positives caused by cross-reactions and batch differences, and significantly improves the detection specificity.
• On the other hand, single base resolution provides the possibility to analyze the site-specific functions of m6A, such as regulatory differences in different regions of mRNA.

More importantly, GLORI 1.0 realized the quantitative analysis of m6A modification for the first time, and accurately reflected the modification abundance by calculating the transformation efficiency (demethylation ratio) of specific sites, which laid a methodological foundation for studying the dynamic regulation mechanism of m6A (such as changes in cell cycle or disease process).

With these characteristics, GLORI 1.0 quickly became the quantitative gold standard in the field, and was widely used to verify the classical m6A regulatory pathway, discover new modification sites, and establish the correlation between modification maps and phenotypes. For example, in tumor research, this technique successfully mapped the m6A difference between cancer cells and normal cells, and revealed the direct correlation between m6A high-abundance modification on oncogene mRNA and translation activation, which provided a new perspective for targeted therapy.

Current transcriptome-wide m6A profiling methods (Tegowski et al., 2024)

Identifying the Challenges: Limitations of GLORI 1.0

Although the technical principle of GLORI 1.0 is revolutionary, the limitations gradually exposed in practical application restrict its wider popularization, mainly in the following three aspects:

High RNA Input Requirements

The requirement of the initial RNA amount of GLORI 1.0 is as high as microgram (usually 5-10μg of total RNA), which makes it difficult to apply it to rare cell populations (such as stem cells and circulating tumor cells) or clinical trace samples (such as free RNA in biopsy tissues and body fluids). Under the background of the rise of single-cell epigenomics, this defect is particularly prominent. The total amount of RNA in single cells is only 10 g, which is far below the detection limit of GLORI 1.0, making it impossible to capture the m6A dynamics in cell heterogeneity.

The treatment of clinical samples highlights this problem: For example, the total amount of free RNA in cerebrospinal fluid of patients with advanced cancer is often less than 1μg, which cannot be effectively detected by traditional GLORI 1.0, which limits the application of this technology in liquid biopsy.

Experimental Process Complexity

The operation process of GLORI 1.0 includes more than ten steps, such as RNA fragmentation, multiple rounds of enzymatic reaction, magnetic bead purification, and library construction, which takes about three days to complete, and many links (such as ALKBH5 enzyme concentration titration and reaction time control) require extremely high operating experience. For example, if the temperature fluctuation of the enzymatic reaction exceeds 1℃, the conversion efficiency may decrease by more than 20%. However, the volume error of magnetic bead purification will significantly affect the RNA recovery rate and introduce technical deviation.

This complexity not only increases the risk of experimental failure but also reduces the comparability of results between different laboratories. A cross-laboratory validation study shows that the coincidence rate of m6A loci reported by different teams is only 65%, which is far below the ideal level (> 80%) when the same sample is detected by GLORI 1.0.

Potential Risk of Incomplete Transformation

The completeness of the enzymatic reaction of ALKBH5 is the core premise of the quantitative accuracy of GLORI 1.0. However, in the actual reaction, m6A may not be completely demethylated due to factors such as the obstruction of RNA secondary structure, the attenuation of enzyme activity, or the lack of cofactors. The untransformed ma locus will be misjudged as "unmodified", resulting in a false negative. More seriously, partial transformation (such as only 50% demethylation) will interfere with the quantitative accuracy and make the calculation of modification abundance biased.

The research shows that the transformation efficiency of GLORI 1.0 can be reduced to below 70% in the RNA region rich in GC (which is easy to form a stem-loop), which is significantly lower than the overall average level (> 90%), which makes the reliability of m6A modification analysis results in these regions questionable.

Block diagram of the GLORI instrument (Motte et al., 2016)

The Genesis of GLORI 2.0: Aims and Design Principles

The development of GLORI 2.0 is not a subversive reconstruction of the original technology, but an accurate upgrade based on the actual application requirements. Its core goal is to break through the bottleneck of sensitivity (reducing RNA input) and practicality (simplifying the process) based on retaining the advantages of high specificity (antibody independence) and single-base resolution of GLORI 1.0, to make it more suitable for the diversified needs of basic research and clinical application. To achieve this goal, the R&D team established three design principles:

Paralleled Performance Retention and Promotion

All optimization measures are based on the premise of not sacrificing detection performance. Specifically, it is necessary to ensure that the specificity (false positive rate < 5%), resolution (single base level), and quantitative accuracy (correlation coefficient > 0.9 with LC-MS/MS detection results) of the upgraded technology are not lower than GLORI 1.0. In the modification of enzyme engineering, it is necessary to screen ALKBH5 mutants by directed evolution, which can not only improve the catalytic activity, but also not reduce the substrate specificity.

The Extremely Simplified Pocess

In view of the tedious steps of GLORI 1.0, the design principle requires that the operation time be shortened by integrating the reaction system, reducing purification steps, and automatic adaptation. The goal is to reduce the whole experiment time to less than one day, and the hands-on time is reduced by more than 50%. At the same time, the dependence on the skills of operators is reduced, making the technology easier to standardize and popularize.

Magnitude Breakthrough in Sensitivity Order

By optimizing the efficiency of the enzymatic reaction, improving the RNA capture method, and improving the sensitivity of library construction, the RNA input was reduced from microgram level to nanogram level (10-100ng), so that it could cover rare sample types (such as RNA of 100-1000 cells). At the same time, it is necessary to ensure the detection repeatability under low input (the correlation coefficient of biological repetition is > 0.85) to avoid data fluctuation caused by insufficient sample size.

The formulation of these design principles provides a clear direction for the technological innovation of GLORI 2.0, ensuring that the upgraded technology can not only solve the actual pain points but also continue the core advantages of the original technology.

dm6 ACRISPR induces demethylation of single m 6 A-modified mRNA (Li et al., 2020)

Overview of Key Innovations in GLORI 2.0

The research and development of GLORI 2.0 technology focuses on the accuracy, sensitivity, and flux requirements of m6A quantitative detection. By integrating multi-disciplinary cutting-edge technologies, it realizes systematic innovation in the dimensions of molecular biology, bioinformatics, and nanotechnology, and successfully achieves the preset research goals. The core breakthrough of this technology is mainly reflected in the following three aspects:

Development of A New Recombinant Enzyme

Through site-directed mutation and directed evolution, the R&D team obtained a highly active and stable mutant (named ALKBH5v2). Compared with the wild-type enzyme, the catalytic efficiency (kcat/Km value) of the mutant was increased by three times, and the half-life at 37℃ was extended from 4 hours to 12 hours, and the substrate specificity of m6A was not affected (the cross-activity to other modifications such as m⁶Am was less than 0.1%).

The advantage of ALKBH5v2 is particularly significant in low input samples: in the 100ng total RNA reaction system, its transformation efficiency can still be maintained above 95%, while the efficiency of the wild-type enzyme is only 65% under the same conditions. This improvement fundamentally solves the risk of incomplete transformation and provides an enzymatic basis for reducing RNA input.

Streamlining and Automation Adaptation of the Process

GLORI 2.0 systematically reconstructs the experimental process:

• Integrated reaction system: RNA fragmentation, enzymatic reaction, and pre-treatment of reverse transcription are integrated into a single buffer liquid system, and three steps of magnetic bead purification are reduced, so that the RNA recovery rate is increased from 60% to over 85%.
• Shorten the reaction time: By increasing the enzyme concentration and optimizing the temperature gradient, the reaction time of ALKBH5v2 was reduced from 60 minutes to 30 minutes, and the transformation efficiency was not affected.
• Automation compatibility: The redesigned reaction system is adapted to the 96-well plate format, and the whole operation can be completed through the liquid workstation, reducing human error and realizing Qualcomm detection (96 samples can be processed at a time).

After the process optimization, the whole experiment time of GLORI 2.0 was shortened from 72 hours to 8 hours, and the hands-on time was reduced from 12 hours to 3 hours, which significantly improved the practicability and standardization of the technology.

Precise Optimization of Reaction Conditions

To meet the detection requirements of low input samples, GLORI 2.0 optimized the reaction conditions with multiple parameters:

Cofactor enhancement: adding a new antioxidant (such as Trolox) to the reaction buffer to maintain the reduction state of Fe²⁺ and keep the enzyme activity stable at low RNA concentration.

• RNA protection strategy: Adding RNase inhibitor mixture (such as Superase-In combined with Murine RNase Inhibitor) can reduce the degradation risk of fragmented RNA, especially suitable for degrading severe clinical samples (such as FFPE tissue RNA).
• Library construction adaptation: A new type of low-deviation linker (such as Tn5 transposon-mediated library construction) is adopted to reduce the preference of PCR amplification and improve the capture efficiency of low-abundance RNA fragments by 2 times.

These optimizations reduced the lower limit of RNA input of GLORI 2.0 to 10ng (total RNA), and still maintained the number of sites detected (about 80% coincidence rate) and quantitative accuracy (Pearson correlation coefficient = 0.89) equivalent to that of GLORI 1.0 (10 μg input) at this level.

m6A detection methods using TGS platforms (Yang et al., 2024)

Conclusion

Based on inheriting the core advantages of GLORI 1.0 (antibody independence, single-base resolution, and quantitative ability), GLORI 2.0 successfully broke through the key limitations of the original technology through enzyme engineering reform, process simplification, and optimization of reaction conditions, and achieved a leap from the gold standard to the practical standard.

This upgrade has greatly expanded its application scenarios: in basic research, it can analyze the m6A modification heterogeneity of rare cell populations (such as neural stem cells and tumor stem cells); In clinical transformation, micro-biopsy samples and free RNA in body fluids can be detected with high accuracy, providing new epigenome markers for disease diagnosis and prognosis evaluation.

In the future, the further development of GLORI 2.0 can focus on two directions: one is to combine with single cell sequencing technology to develop single cell GLORI-seq to reveal the m6A dynamics at the individual level; The second is to expand to the detection of other RNA modifications (such as MC and ψ), and to construct a panoramic epigenome analysis platform by replacing specific enzymes (such as MC demethylase).

In a word, the birth of GLORI 2.0 not only improved the practicability of m6A detection technology but also promoted the leap of epigenomics from batch sample research to micro, single-cell, and even clinical sample research, which injected new impetus into the basic and transformation research in this field.

References

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