Application and Case Studies Sharing of bDNA Technology for Oligonucleotide and mRNA Drug Bioanalysis

With the continuous advancement of technology, Biomedicineis booming, and drug forms vary greatly, extending from traditional chemicals, antibodies, proteins, and peptides to cells, nucleic acids, and other forms.  The continuous emergence of new drug forms also poses some high requirements and new challenges to drug related bioanalytical technologies, and new analytical technologies are constantly being introduced.  Medicilon New Media’s bioanalysis series column continues to invite senior professionals in the field to talk and discuss about bioanalysis from different perspectives.  In this issue, we will share a different analysis technology for nucleic acid drugs: bDNA.

In recent years, nucleic acid drugs have ushered in a stage of rapid development.  Since 2016, a total of 15 nucleic acid drugs have been successfully approved for marketing in the United States and/or the European Union, including 13 oligonucleotide drugs and 2 mRNA vaccines.  Among them, 8 of the oligonucleotide drugs are antisense oligonucleotide (ASO), and 5 are small interfering RNA (siRNA).  It can be seen that oligonucleotides and mRNA represented by ASO and siRNA have become popular subdivisions in the research and development of nucleic acid drugs.  More and more oligonucleotides and mRNA have entered the IND or clinical phaseworldwide.

In addition to the common problems that need to be considered and overcome such as synthetic modification, delivery, stability and immunogenicity, nucleic acid drugs cannot avoid the challenges of pharmacokinetic research when entering the IND and clinical stages.  Pharmacokinetic bioanalytical methods are a very important technical challenge in practice that we have to face.  For mRNA, it is easily degraded by nucleases, unstable, and has a short half-life.  The bioabalytical method needs to be highly sensitive. Although RT-qPCR is highly sensitive and can perform quantitative analysis for mRNA, it involves complex nucleic acid extraction and reverse transcription.  Moreover, there will be problems such as losses during the nucleic acid extraction process and the absolute extraction recovery rate is uncontrollable.  Although the instability and short half-life of oligonucleotides can be improved through modification and other technical means, their molecular weight is neither large nor small.  (Generally, ASO is a single-stranded form of 18-30nt, and siRNA is a double-stranded form of 20-25nt)  Whether it is traditional small molecule analysis technology mass spectrometry or traditional nucleic acid analysis technology qPCR, their analysis is challenging. For example, mass spectrometry technology involves residue, retention, instrument contamination and sensitivity issues.  Although the Stem-loop RT-qPCR method can be barely used for oligonucleotides test, the qPCR technology itself is more suitable for longer nucleic acid molecules and is less suitable for modified nucleic acid molecules in practice.

In addition to mass spectrometry and qPCR technology, liquid phase fluorescence detection technology and Hybrid-ELISA (hELISA) technology are also used in the analysis of nucleic acid drugs.  Each of these methods has its own advantages and disadvantages respectively.  Some of them require a higher sample volume, some methods are still not sensitive enough, and some involve complex reaction steps or the use of special enzymes and other limitations.  There is currently another nucleic acid analysis technology, bDNA (branch DNA) technology, which can make up for some of the shortcomings of the above technologies to varying degrees.

1. bDNA Technology and Advantages

bDNA technology is branch DNA technology, which is one analytical technology integrating molecule labeling, probe design, molecule hybridization, fluorescence or chemiluminescence detection.  The essence of this technology is an upgraded version of nucleic acid molecule hybridization technology, which also requires specific capture probes and detection probes.

However, both the capture probe and the detection probe in this technology have additional extension sequences.  The capture probe can use its extension sequence to complementarily bind to the pre-coated universal capture sequence on the Assay Plate.  The detection probe has a double “Z”-shaped special structure, and its lower end is specifically complementary to the target molecule.  The extended part of the upper end of the “Z”-shaped probe can be combined with the pre-amplification probe, and then the pre-amplification probe, amplification probe, and labeling probe step by step to form a probe set with a dendritic structure to achieve cascade amplification of the detection signal, thereby achieving the effect of improving sensitivity (Fig. 1). For shorter small nucleic acids, the double “Z” – shaped structure of the detection probe can be flexibly replaced by non “Z” – shaped extended probes, as long as the extended sequence can complementarily bind to the pre-amplified probe.

Fig. 1 Schematic diagram of bDNA technology process and principle

However, both the capture probe and the detection probe in this technology have additional extension sequences.  The capture probe can use its extension sequence to complementarily bind to the pre-coated universal capture sequence on the Assay Plate.  The detection probe has a double “Z”-shaped special structure, and its lower end is specifically complementary to the target molecule.  The extended part of the upper end of the “Z”-shaped probe can be combined with the pre-amplification probe, and then the pre-amplification probe, amplification probe, and labeling probe step by step to form a probe set with a dendritic structure to achieve cascade amplification of the detection signal, thereby achieving the effect of improving sensitivity (Fig. 1). For shorter small nucleic acids, the double “Z” – shaped structure of the detection probe can be flexibly replaced by non “Z” – shaped extended probes, as long as the extended sequence can complementarily bind to the pre-amplified probe.

bDNA technology has been used in the fields of cosmetics R&D, biopharmaceutical and virus detection. This technology is widely used in overseas pharmaceutical R&D, but currently there are few practical applications in China pharmaceutical R&D field.  Due to its high sensitivity and low sample consumption, it has unique application advantages in preclinical pharmacokinetics and tissue distribution studies, especially for those small animal experiments with special administration, special materials, and limited sampling volume.  This technology can be widely used in the detection of various oligonucleotides such as ASO, siRNA, and mRNA.  Moderna has used this technology to conduct its mRNA biodistribution studies.

Medicilon Biotechnology Drug Analysis Department has not only successfully applied this technology to the analysis of mRNA products, but also successfully applied it to the analysis of the two most popular oligonucleotide drugs, ASO and siRNA, which is the first in China to realize the comprehensive application of this technology in nucleic acid drugs.  This article will briefly introduce the application of this technology based on the actual case studies.

2. Application of bDNA Technology for mRNA Bioanalysis

mRNA is a single-stranded nucleic acid molecule with larger length and  more number of bases than ASO and siRNA.  Therefore, , bDNA has significant advantages compred to oligonucleotides when it is used for mRNA.  Because the mRNA is long enough, multiple pairs of double “Z” probes can be designed for one mRNA molecule. The more Z-shaped probes, the higher the signal amplification and the easier it is to improve the sensitivity.

It is also necessary to design some blocking probes that are complementary to the mRNA to prevent non-specific signals in addition to designing specific capture probes and double “Z” probes based on the mRNA sequence when the using of this technology for mRNA and, which is different from the utilization for ASO and siRNA (Fig. 2).

Fig. 2 Schematic diagram of the principle of bDNA technology in mRNA analysis

Based on the principle that bDNA technology can be well applied to mRNA analysis, we have developed an analysis method for tissue distribution of a certain mRNA molecule using bDNA technology.  As shown in the example below, we can use bDNA technology to not only reduce the detection limit of mRNA in tissues to less than 100 copies/uL, but also its accuracy and precision can even fully meet the acceptance standards required by regulations for LBA technology.

It can be seen from the standard curve that in the bDNA technology method, the relationship between the doubling of the instrument response signal and the doubling of the concentration of standards of different concentrations is close to the ideal direct proportional relationship (Table 1), so linear fitting can be achieved using a linear equation (Fig. 3).  Because it breaks away from the utilization of the antigen-antibody reaction relationship, the relationship between instrument response signal and concentration for bDNA is very different from LBA technology and hybrid-ELISA technology, the former（bDNA） can be fitted in a straight line, while the latter two (LBA and hybrid-ELISA) require curve fittingwith 4-parameter or 5-parameter.

Table 1 Standard curve data for bDNA detection of one mRNA

Fig. 3 Standard curve fitting for bDNA detection of one mRNA

In a preclinical study, only a very small part of the tissue samples in the first round of sample analysis required re-analysis, and the repeated results were almost all consistent with the results from the initial analysis, that is, more than 90% of the samples were consistent with the results of the initial analysis (Fig. 4).  Data shows that the method for bDNA technology has good reproducibility.

Fig. 4 Reproducibility of bDNA method for one mRNA analysis method

3. Application of bDNA for Antisense Oligonucleotide (ASO) Bionalysis

The application of bDNA to ASO and siRNA is different from its application to mRNA, because the former two are oligonucleotides, and the chain is very short, so it is not necessary to design blocking probe, and it is also inconvenient to use double “Z” type probes because of the too short chain.  It is also even more impossible to use multiple double “Z”-shaped probes to amplify signals. Generally, only non-“Z”-shaped sequence extension probes can be used as shown in (Fig. 5).  Therefore, the analysis of ASO is relatively more difficult than that of mRNA.

Fig. 5 Schematic diagram of the ASO principle of bDNA technology analysis

Medicilon Biotechnology Drug Analysis Department has also applied bDNA to the analysis of ASO in plasma, and achieved satisfactory sensitivity (see Table 2 and Fig. 6).  Although the process of developing bDNA Assay and exploring reaction conditions is more difficult and complicated than that of mRNA, the reaction steps and reaction systems need to be flexibly changed for different ASOs based on the experience of this laboratory.  Although commercial kits containing universal probes, buffers and substrates are currently available, the process and approach of establishing the method cannot be limited by the reagents provied in the kit.

In order to adapt to the characteristics of the specific ASO in intended study, especially to avoid the influence from stability and matrix interference, we need to specifically explore and replace some special buffer or lysis buffer components, whose performance cannot be provided by general commercial reagents.  In terms of achieving and improving sensitivity, we basically believe that bDNA technology is relatively easier to improve assay sensitivity than Hybrid-immnoassays for the same small nucleic acid molecule,.

Table 2 Standard curve data for bDNA detection of one ASO（ASO-X）

Fig. 6 Standard curve fitting for bDNA detection of one ASO（ASO-X）

4. Application of bDNA for Small Interfering RNA (siRNA) Bioanalysis

The analysis of siRNA based on bDNA technology essentially involves constructing a specific probe for one of the strands and performing signal amplification. The reaction schematic diagram is similar to ASO, so  Schematic diagram is not shown in this article separately, but readers can still refer to Fig. 5.  However, there are also significant differences in how bDNA is used to construct ASO and siRNA assays.  ASO is a single-stranded structure, while siRNA is a double-stranded structure. Therefore, the application of bDNA technology to ASO is easier to implement than that of siRNA.  siRNA is relatively challenging when constructing analysis methods using bDNA technology platforms. Because of the double-stranded characteristics of siRNA itself, an additional denaturation step is required for siRNA samples.  After denaturation, it is necessary to prevent the complementary strand from renaturing itself during annealing and hybridization, which affects the binding of the capture probe and detection probe to the target strand. This is the biggest concern unlike single-stranded RNA.

The selection of probe sequence and exploration of annealing incubation temperature are very important.  In addition, the influence of stability and different matrices cannot be avoided, and the exploration of various siRNA reaction conditions in method development is more complicated.  These are important challenges that need to be overcome in the practice of applying this technology to siRNA analysis.

Once these basic issues are addressed, high sensitive siRNA analysis methods can also be developed successfully.  The chart below shows a case study of Medicilon Laboratory analyzing one siRNA in plasma based on bDNA technology.  As shown in Table 3, pg/mL level sensitivity can be achieved easily.  Whether it is ULOQ (80pg/mL) or LLOQ (1.25pg/mL), different sets of quality control samples at each concentration can reach the recovery acceptance range as that of the traditional PK analysis method (Fig. 7).  The instrument response value at this sensitivity is relatively low but can still perform a good fitting with a linear equation (Fig. 8).

Table 3 Standard curve data of bDNA detection for one siRNA(siRNA-X)

Fig. 7 Recovery rates of 3 sets QCs at different levels

Fig. 8 Standard curve fitting for bDNA detection of one siRNA (siRNA-X)

5. Summary and Prospect

For small oligonucleotides fields, in addition to being used in ASO and siRNA, bDNA can also be used in various small nucleic acid molecules such as miRNA and nucleic acid aptamers.  The application principles and forms are generally similar to the previous two, and there are currently no official drugs for miRNA and nucleic acid aptamers (one was withdrawn after being marketed), so they will not be described in detail here.

In summary, bDNA technology is a technology that can be widely used in various types of nucleic acid bioanalysis, whether it is long mRNA or short small oligonucleotides.  Compared with a series of technologies such as LC-MS/MS or HRMS and Hybrid-immunoassays, it is relatively easier to improve assay sensitivity.  At present, in the field of gene therapy, more and more nucleic acid drugs use special local administration methods, which often involve special materials such as cerebrospinal fluid, small animal urine, and even some biopsy tissue samples.  Therefore, related bioanalysis has also put forward a higher practical demand for high sensitivity and low sample consumption, which is also a realistic demand for relevant medical researchers.  bDNA is a very suitable technology to meet such needs.

For mRNA bioanalysis, although the traditional RT-qPCR method can be analyzed and has high sensitivity,  it is impossible to avoid the absolute loss of nucleic acids during the extraction process, and there will be variation among different extraction reagents, personnel, or extraction instruments because the nucleic acid extraction steps are complicated.  This may potentially discount the actual sensitivity of the qPCR method.  bDNA technology avoids the impact of these sample handling processes, and can use the copy number of the target analyte per unit volume as its absolute quantitative unit. Moreover, once bDNA technology methods are developed, they are much more robust than qPCR methods. Traditional industry technical acceptance standards for Pharmacokinetic bioanalysis can also be used to constrain the quality control of such methods，which is a significant difference from qPCR technology.  

Based on the comprehensive application and experience of Medicilon Bioanalytical Laboratory, bDNA technology is undoubtedly a nucleic acid detection technology with significant advantages and application value suitable for the pharmaceutical research and development.

Although bDNA is a relatively superior technology, its actual application in the analysis of various nucleic acid molecules cannot be accomplished at one stroke. It also requires a more complex method development and optimization process.  Like Hybrid-immunoassays, this type of technical method highly relies on the design of specific probes to achieve assay specificity.  Even though all types of probes are available, it still takes a long time of efforts to explore the reaction conditions and formulas of various reaction buffers and even by adjusting a series of conditions such as the reaction modes, reaction systems, and reaction sequences of different probes so that one satisfactory and applicable assay method can be developed and established.

As mentioned above, bDNA is applicable in various types of nucleic acid analysis.  For mRNA, single-stranded oligonucleotides (ASO) and double-stranded oligonucleotides (siRNA), the application difficulty is progressive, and the bDNA method for specific drug molecules in each type of nucleic acid is also personalized.  The difficulty varies depending on the molecule itself, requiring the development of analytical methods specific to the sequence and characteristics of the target molecule.  In the development of bioanalytical methods, bDNA technology does not significantly save time and energy compared with other types of methods. Moreover, bDNA technology has a higher application cost than other nucleic acid analysis technologies due to the specific structure of its probes.  However, once a method based on the principles of bDNA technology is successfully developed, it is very reliable with better robustness and reproducibility.

bDNA technology can also perform multiplex analysis. The double “Z” probes or other extension probes and signal amplification systems involved can not only be used directly for nucleic acid detection, but can also be expanded to other analysis and detection platforms.  Thermo has applies this type of probe technology to flow cytometry platform for transcript detection at the cellular level.  Fixed and ruptured cells can be directly incubated with specific extended labeled probes for signal amplification based on signal amplification system, and finally detected by flow cytometry,which is called PrimeFlow RNA analysis technology.  In addition to expanding applications in streaming platforms, the double “Z” type probe and signal amplification system can also be applied by RNAscope technology for in situ hybridization of target RNA in tissue cells.  This makes RNA in situ hybridization highly specific, improves the sensitivity of single-molecule detection and brings an extremely high signal-to-noise ratio, and can simultaneously quantify the expression of multiple RNAs at the single cell level.  Obtaining single-copy RNA expression data in single cells while providing complete tissue morphology information is also a technology demand in the field of small nucleic acid research and development to evaluate drug distribution based on histology.

Although there are relatively few applications of bDNA technology in Chinese pharmaceutical research and development field currently , with the vigorous development of gene therapy drugs, especially nucleic acid drugs in its branch field, bDNA technology will be familiar and favored by a large number of biomedicine researchers, and the performance of this technology will be recognized by more and more scientists.