Case Studies and Publications

Secondary Structure and Stability of Imperfect G-Quadruplex Oligonucleotides

G-quadruplexes (GQs) are important biomarkers and drug targets because of their regulatory role in transcription. Adherence to the GQ consensus sequence is often used to predict new GQ regions, but this approach can overlook potential candidates that form imperfect G-Quadruplex (imGQs) structures containing bulges, vacancies or mismatches.

Multiple imGQs were characterized with regards to secondary structure, thermal stability and interaction with small molecule ligands. By making use of CD spectroscopy and orthogonal absorbance and fluorescence data obtained with a Chirascan, imGQs were shown to behave similarly to perfect GQs, suggesting that GQs with potential regulatory function are more abundant than commonly assumed.

Rational Optimization of Solvent Conditions for G-Quadruplex Oligonucleotides

As the regulatory role of G-quadruplex structures in crucial biological processes emerges, CD spectroscopy can be expected to gain increased importance in their characterization as drug targets for anti-viral and anticancer therapies.

In this study, CD spectroscopy was used as an orthogonal technique to optimize experimental conditions for Mass Spectrometry experiments, which can be used for high-throughput screening in drug development with regards to structural analysis and binding interactions.


Follow the Formation of Organometallic Complexes

(Selegård et al., 2017)

Follow the Formation of Organometallic Complexes

Using a Chirascan CD Spectrometer, it was found that the addition of zinc ions to peptide-conjugated hyaluronic acid triggered folding of the peptides into a helix-loop-helix motif and dimerization into four-helix bundles, resulting in hydrogelation of the hybrid polymer.

See the full study here.

Determine Binding Affinities of Ligands

(Hughes et al., 2017)

Determine Binding Affinities of Ligands

The binding constants (kd) for the binding between a protein that confers antibiotic resistance and two glycopeptide antibiotics (vancomycin and teicoplanin) were determined using Chirascan CD spectroscopy. The two antibiotics were found to bind with different affinities and mechanisms.


See the full study here.

Compare Tertiary Structures of Protein Homologues

(Zuberek and Stelmachowska., 2017)

Compare Tertiary Structures of Protein Homologues

Near-UV Circular Dichroism spectra performed on a Chirascan revealed a reverse orientation of a tryptophan residue important for cap-binding of mRNA in a key factor for translation initiation in yeast as compared to the human homologue.


See the full study here.

Reveal the stereoconfiguration of small molecules

(Kries et al., 2017)

Reveal the stereoconfiguration of small molecules

CD analysis of the natural reaction products of two enzyme homologues (CrISY and AmISY) from different species of flowering plants revealed opposite stereoconfigurations when performed on a Chirascan V100.


See the full study here.

Comparison of native and stressed mAb

(Data courtesy of a biotherapeutic development company)

Spectra normalized for protein concentration by simultaneous absorbance measurements, 0.5 mm pathlength, Chirascan™ V100

Gain insight into secondary structure

Typical beta-sheet structure retained in native and stressed mAb.

Disulphide bond environment unchanged.

Spectra normalized for protein concentration by simultaneous absorbance measurements, 10 mm pathlength, Chirascan™ V100

Reveal tertiary structure

Environment of aromatic amino acid side-chains appears similar.

Note: A subsequent HOS comparison study using a Chirascan™ Q100 system confirmed that minor differences in tertiary structure were statistically significant (data not shown).


Review of spectra suggests no difference between native and stressed mAb.

Effect of transient exposure to nanoparticles

(Data courtesy of a leading research university, Germany)

Spectra normalized for protein concentration by simultaneous absorbance measurements, 0.5 mm pathlength, Chirascan™ V100

Determine structural effects

Significant change in far UV CD spectrum of an enzyme indicated perturbation of secondary structure by nanoparticle exposure.

Continuous temperature ramp 20°- 90°C, 71 spectra in 71 min, 1°C/min, 0.8 sec per point, 1 nm bandwidth, 1 nm step, Chirascan™ V100

Determine effect on protein stability

Thermal denaturation curves showed altered folding profile after nanoparticle exposure. Global fit of multiwavelength data confirmed a 2.9°C change in melting temperature.

Global fit of multiwavelength thermal denaturation data


Exposure to nanoparticles altered folding of a globular protein which, in turn, enhanced protein stability.


Publications Featuring Chirascan Instruments

Since the introduction of the first instrument in 2005, Chirascan CD spectrometers have contributed to thousands of peer-reviewed publications. Below is a small selection of the most recent publications showing the breadth of applications.


Austerberry et al., 2017. “The Effect of Charge Mutations on the Stability and Aggregation of a Human Single Chain Fv Fragment.” European Journal of Pharmaceutics and Biopharmaceutics 115 (June): 18–30.

Berdnikova et al.2017. “Governing the DNA-Binding Mode of Styryl Dyes by the Length of Their Alkyl Substituents – From Intercalation to Major Groove Binding.” Organic & Biomolecular Chemistry.

Bönisch et al., 2017. “Novel CH1:CL Interfaces That Enhance Correct Light Chain Pairing in Heterodimeric Bispecific Antibodies.” Bispecific Antibodies, Mutants. Protein Engineering, Design and Selection 287: 1–12.

Bui and Nguyen., 2016. “Insights Into the Interaction of CD4 with Anti-CD4 Antibodies.” Immunobiology 222 (2): 148–54.

Elmer-Dixon and Bowler. 2017. “Site A-Mediated Partial Unfolding of Cytochrome c on Cardiolipin Vesicles Is Species Dependent and Does Not Require Lys72.” Biochemistry, August, acs.biochem.7b00694.

Grunér et al., 2017. “The Dynamics of Multimer Formation of the Amphiphilic Hydrophobin Protein HFBII.” Stopped-Flow, Fluorescence, Multimerization. Colloids and Surfaces B: Biointerfaces 155: 111–17.

Hickey et al.,2018. “Analytical Comparability Assessments of Five Recombinant CRM 197 Proteins from Different Manufacturers and Expression Systems.” Journal of Pharmaceutical Sciences, March. 

Lei and Bowler2018. “Humanlike Substitutions to Ω-Loop D of Yeast Iso-1-Cytochrome c Only Modestly Affect Dynamics and Peroxidase Activity.” Journal of Inorganic Biochemistry, February. 

Liu et al., 2017. “Ratiometric Fluorescence Sensor for the microRNA Determination by Catalyzed Hairpin Assembly.” ACS Sensors, September. 

Matukumalli, Tangirala, and Rao. 2017. “Clusterin: Full-Length Protein and One of Its Chains Show Opposing Effects on Cellular Lipid Accumulation.” Scientific Reports 7 (January). Nature Publishing Group: 41235.

Miao et al., 2017. “Physicochemical and Biological Characterization of the Proposed Biosimilar Tocilizumab.” BioMed Research International 2017 (March). Hindawi: 1–13.

Moreno et al., 2016. “Study of Stability and Biophysical Characterization of Ranibizumab and Aflibercept.” European Journal of Pharmaceutics and Biopharmaceutics 108: 156–67.

Narang, Singh, and Mukhopadhyay. 2017. “Stepwise Unfolding of Human β2-Microglobulin Into a Disordered Amyloidogenic Precursor at Low pH.” European Biophysics Journal 46 (1). Springer Berlin Heidelberg: 65–76.

Nejat Dehkordi and Akerman. 2018. “Interaction of DNA with Water Soluble Complex of Nickle and Formation of DNA Cross-Links.” Chemico-Biological Interactions, January.

Nwaji, Mack, and Nyokong. 2018. “Photophysical and Strong Optical Limiting Properties of Ball-Type Phthalocyanines Dimers and Their Monomeric Analogues.” Journal of Photochemistry and Photobiology A: Chemistry 352 (February): 73–85.

Raasakka et al., 2015. “Determinants of Ligand Binding and Catalytic Activity in the Myelin Enzyme 2′,3′-Cyclic Nucleotide 3′-Phosphodiesterase.” Scientific Reports 5: 16520. 

Roeters et al., 2017. “Evidence for Intramolecular Antiparallel Beta-Sheet Structure in Alpha-Synuclein Fibrils from a Combination of Two-Dimensional Infrared Spectroscopy and Atomic Force Microscopy.” Amyloid Fibrils, Intrinsically Disordered Proteins. Scientific Reports 7: 41051.

Ropiak et al., 2017. “Identification of Structural Features of Condensed Tannins That Affect Protein Aggregation.” Edited by Jamshidkhan Chamani. PLOS ONE 12 (1): e0170768.

Roth et al., 2018. “Functionalized Calcium Carbonate Microparticles for the Delivery of Proteins.” Nanoparticle Interactions, Stability. European Journal of Pharmaceutics and Biopharmaceutics 122: 96–103.

Sani et al., 2017. “One Pathogen Two Stones: Are Australian Tree Frog Antimicrobial Peptides Synergistic against Human Pathogens?” European Biophysics Journal, May, 1–8.

Singh et al., 2017. “Effect of Polysorbate 20 and Polysorbate 80 on the Higher-Order Structure of a Monoclonal Antibody and Its Fab and Fc Fragments Probed Using 2D Nuclear Magnetic Resonance Spectroscopy.” Journal of Pharmaceutical Sciences 106: 3486–98.

Slator et al., 2018. “Di-Copper Metallodrugs Promote NCI-60 Chemotherapy via Singlet Oxygen and Superoxide Production with Tandem TA/TA and AT/AT Oligonucleotide Discrimination.” Synthetic Co-Polymers, Metallodrugs. Nucleic Acids Research.

Townsend et al., 2017. “Heparin and Methionine Oxidation Promote the Formation of Apolipoprotein A-I Amyloid Comprising α-Helical and β-Sheet Structures.” Biochemistry 56 (11). American Chemical Society: 1632–44.

Varizhuk et al., 2017. “The Expanding Repertoire of G4 DNA Structures.” G-Quadruplexes, Melting. Biochimie 135: 54–62.

Vorob’ev et al., 2017. “Physicochemical Properties, Toxicity, and Specific Activity of a Follitropin Alpha Biosimilar.” Pharmaceutical Chemistry Journal 50 (11). Springer US: 753–60.

Wei et al., 2018. “Polyoxygenated Cyathane Diterpenoids from the Mushroom Cyathus Africanus, and Their Neurotrophic and Anti-Neuroinflammatory Activities.” Natural Isolates, Stereoconfiguration. Scientific Reports 8: 2175.

Y. Wei et al., 2017. “Effect of Phosphate Ion on the Structure of Lumazine Synthase, an Antigen Presentation System from Bacillus Anthracis.” Journal of Pharmaceutical Sciences 0.

Yang et al., 2017. “A Novel Chimeric Lysin with Robust Antibacterial Activity against Planktonic and Biofilm Methicillin-Resistant Staphylococcus Aureus.” G-Quadruplexes, Ligand Binding. Scientific Reports 7: 40182.

Zhao et al., 2016. “Structural Insights on PHA Binding Protein PhaP from Aeromonas Hydrophila.” Scientific Reports 6 (1): 39424.

Zhao et al., 2017. “Modelling of Microbial Polyhydroxyalkanoate Surface Binding Protein PhaP for Rational Mutagenesis.” Microbial Biotechnology 10: 1400–1411.

Further information