Aptamer selection by direct microfluidic recovery and surface plasmon resonance evaluation.

A surface plasmon resonance (SPR)-based SELEX approach has been used to raise RNA aptamers against a structured RNA, derived from XBP1 pre-mRNA, that folds as two contiguous hairpins. Thanks to the design of the internal microfluidic cartridge of the instrument, the selection was performed during the dissociation phase of the SPR analysis by recovering the aptamer candidates directly from the target immobilized onto the sensor chip surface. The evaluation of the pools was performed by SPR, simultaneously, during the association phase, each time the amplified and transcribed candidates were injected over the immobilized target.

Extending ITC to Kinetics with kinITC.

Isothermal titration calorimetry (ITC) has long been used for kinetic studies in chemistry, but this remained confined to enzymatic studies in the biological field. In fact, the biological community has long had the tendency of ignoring the kinetic possibilities of ITC considering it solely as a thermodynamic technique, whereas surface plasmon resonance is seen as the kinetic technique par excellence. However, the primary signal recorded by ITC is a heat power which is directly related to the kinetics of the reaction.

Mutations of the SL2 dimerization sequence of the hepatitis C genome abrogate viral replication.

Stem-loop SL2 is a self-interacting palindromic sequence that has been identified within the hepatitis C virus genome (HCV). While, RNA dimerization of the HCV genome has been observed in vitro with short RNA sequences, the role of a putative RNA dimerization during viral replication has not been elucidated. To determine the effect of genomic dimerization on viral replication, we introduced mutations into SL2 predicted to disrupt genomic dimerization.

Simulated single-cycle kinetics improves the design of surface plasmon resonance assays.

Instruments based on the surface plasmon resonance (SPR) principle are widely used to monitor in real time molecular interactions between a partner, immobilized on a sensor chip surface and another one injected in a continuous flow of buffer. In a classical SPR experiment, several cycles of binding and regeneration of the surface are performed in order to determine the rate and the equilibrium constants of the reaction. In 2006, Karlsson and co-workers introduced a new method named single-cycle kinetics (SCK) to perform SPR assays.

Direct evidence for RNA-RNA interactions at the 3' end of the Hepatitis C virus genome using surface plasmon resonance.

Surface plasmon resonance was used to investigate two previously described interactions analyzed by reverse genetics and complementation mutation experiments, involving 5BSL3.2, a stem-loop located in the NS5B coding region of HCV. 5BSL3.

Single-cycle kinetic analysis of ternary DNA complexes by surface plasmon resonance on a decaying surface.

Complexes involving three DNA strands were used to demonstrate that the single-cycle kinetics (SCK) method, which consists in injecting sequentially samples at increasing concentrations and until now used exclusively to investigate bimolecular complexes by surface plasmon resonance, can be extended to the kinetic analysis of ternary complexes. DNA targets, B, were designed with sequences of variable lengths on their 3' sides that recognise a surface-immobilized biotinylated DNA anchor, A. These targets displayed on their 5' sides sequences that recognise DNA oligonucleotides of variable lengths, C, namely the analytes.