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Far-UV CD Analysis During Biotherapeutic Development: A Method Capability Study

In this study carried out by Novartis, far- UV CD spectroscopy was qualified for the early development of a monoclonal antibody therapeutic. The high sensitivity of Chirascan V100 enabled low-level detection of an lgG1 mAb in mixture with a structurally high similar mAb. A demountable short-pathlength cell was used to minimize spectral contributions by formulation buffer excipients. Limit of Detection and Limit of Quantification was established, and CD data contributed to a successful biotherapeutic submission to regulatory authorities.

 

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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.

 

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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.

 

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Determination of Enzyme Kinetics Using Stopped-Flow Spectroscopy

This study describes the use of an SX20 spectrometer to measure the presteady state kinetics of a well-studied enzymatic reaction: hydrolysis of p-Nitrophenyl acetate catalysed by α-chymotrypsin. Kinetic parameters such as rate constants and the Michaelis-Menten constant are evaluated.

Since it was first studied over 50 years ago, extensive research has established the detailed mechanism of this reaction.

 

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Stopped-Flow Fluorescence Polarisation/Anisotropy

Stopped-flow fluorescence polarisation/anisotropy is a highly useful technique for obtaining valuable kinetic information about biomolecular interactions and structural changes of biomolecules. An example from the literature illustrates how the SX20 Fluorescence Polarisation accessory is used to gain insights into the kinetics of the interaction between a helicase and its RNA binding partner.

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Stopped-Flow Fluorescence (Förster) Resonance Energy Transfer (FRET)

Stopped-flow FRET is a useful technique for studying many types of biomolecular interactions. The method provides kinetic information regarding distance changes occurring on the millisecond timescale. This application note gives an introduction to the technique along with a review of some application experiments from the scientific literature.

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High Sensitivity Direct Mount Photodiode Array for the SX20

The directly mounted photodiode array accessory ensures highly efficient light collection for stopped-flow absorbance measurements. The improved design ensures that ultimate sensitivity is achieved even at levels that maybe needed to study species that exhibit photobleaching. As an example of the many applications of this accessory, this application note demonstrates its use to measure the kinetics of an inorganic ligand exchange reaction.

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