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Nucleic Acid Analytical Characterization

Experienced Nucleic Acid Therapeutic Characterization

Gain insights about your nucleic-acid derived therapeutic’s safety, efficacy, and developability with stage-appropriate analytical and biophysical characterization. Our team’s experience ranges from small nucleic drugs and highly modified oligonucleotides (e.g., ASO, siRNA, PMO/PPMO, PNA) to larger RNA and DNA molecules needed for gene editing and gene therapies.

Reliable analytical characterization is crucial for informing your molecule’s downstream development. However, nucleic acid-specific factors such as polarity and instability make this challenging. Together with our team you can overcome these obstacles and power your project’s analytical, formulation, and process development needs.

Whether you need routine testing or custom services and advanced characterization studies, we are ready to help advance your program.

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Analytical Characterization

Identity Methods

Mass spectrometry (LC-MS, LC-MS/MS) is used to identify synthetic oligonucleotides (e.g., ASO, siRNA, gRNA), with supporting techniques such as melting temperature analysis also used for oligonucleotides with higher order structure (e.g., siRNA, tRNA). Our team confirms the identity of larger DNA and RNA molecules with sequence mapping (LC-MS, LC-MS/MS), sequencing (e.g., next generation, Sanger), and primer-based techniques (e.g., PCR, RT-PCR).

Purity Methods

Profiling your nucleic acid therapeutic’s purity requires a strategic combination of approaches. Anion exchange (AEX) and ion-pair reversed-phase (IPRP) chromatography (HPLC, U(H)PLC) are commonly used to determine the purity of synthetic oligonucleotides as well as larger RNA and DNA. Additionally, size exclusion chromatography (SEC) is used for synthetic oligonucleotides (siRNA) as well as medium-sized RNA (e.g., gRNA, tRNA) where folding is expected and can significantly affect the size. For RNA and DNA, our team leverages electrophoresis (e.g., CGE, agarose, PAGE), which on occasion, is used for synthetic oligonucleotides, as well.

The integrity of the 5’-capping dinucleotides and the length of the Poly A tail are also critical quality attributes that need to be controlled for linear mRNA therapeutics. We use CGE, IPRP, or LC-MS approaches to characterize the mRNA termini following selective hydrolysis with nucleases to obtain smaller molecules amenable to these analytical techniques.

Nucleic Acids Conformation

Nucleic acids can self-assemble through intramolecular base pairing to form complex secondary and tertiary structures. We determine the size and volume of RNA or DNA molecules with dynamic light scattering (DLS) or SEC with multiangle light scattering (SEC-MALS). 

Our team also assesses nucleic acids conformation in different solutions and temperatures with circular dichroism (CD) spectroscopy. Additionally, UV/VIS or CD spectroscopy are used to study the thermal stability of the structure in nucleic acids. To examine the structure of synthetic oligonucleotides, we use NMR spectroscopy, but this approach is most often used only for characterization.

Molar Extinction Coefficient

Synthetic oligonucleotides are hygroscopic and generally synthesized as sodium salts, meaning that you must determine the water (KF) and sodium content (ICP-OES, ICP-MS) to get a reliable extinction coefficient. Since higher order structure is more common with DNA and RNA molecules, our expert team has developed approaches to control the influence of structure when determining extinction coefficients.

Assay Methods

Spectroscopy (UV/Vis) is the most common approach to determine the concentration of your nucleic acids. Still, the nucleic acid’s structure (e.g., hybridization, base-stacking), affects the extinction coefficient of all nucleic acids and requires careful selection of diluents or sample processing. Assay methods for DNA and RNA can also use RT-qPCR/RT-dPCR or qPCR/dPCR.


In addition to the compendial testing for endotoxin, bioburden, sterility, and elemental impurities for all nucleic acid drug substances, we also detect double-strand RNA (dsRNA) using Immunoblot or ELISA methods.

Physiochemical Characterization

Synthetic oligonucleotides that are manufactured as lyophilized powders and may need additional characterization (e.g., Reference Standard). We can confirm the lyophilized powders are amorphous (XRPD) and perform Differential Scanning Calorimetry (DSC) and Thermogravimetric Analysis (TGA) to characterize the solid material further.

Drug Product Characterization

We provide a suite of additional analytical characterization services suited for programs that have advanced into a drug product. Learn more below.

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Integrated Laboratory Services  

Characterization insights apply across the development timeline, and our experts are ready to sync up with your project. Discover additional opportunities for us to partner with you.

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