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Affinity enhancement bivalent morpholino for pretargeting: initial evidence by surface plasmon resonance
Jiang He1, Guozheng Liu, Jean-Luc Vanderheyden
1Division of Nuclear Medicine, Department of Radiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, Massachusetts 01655, USA. jiang.he@umassmed.edu
Bioconjugate Chemistry
|March 17, 2005
Summary
A novel bivalent Morpholino (MORF) was synthesized and demonstrated superior binding affinity compared to its monovalent form. This enhanced binding, confirmed by surface plasmon resonance, suggests bivalent MORFs offer advantages for pretargeting applications.
Area of Science:
- Bioconjugation chemistry
- Molecular recognition
- Oligonucleotide analogs
Background:
- Pretargeting strategies enhance therapeutic efficacy by improving drug delivery and targeting.
- Bivalent effectors, like Morpholinos (MORFs), offer potential for superior affinity through avidity effects.
- Adjustable linker lengths in MORFs allow for optimization of binding interactions.
Purpose of the Study:
- To synthesize a bivalent MORF and evaluate its binding characteristics.
- To compare the in vitro hybridization affinity of bivalent MORF against its monovalent counterpart.
- To determine if bivalent MORF exhibits bimolecular binding using surface plasmon resonance (SPR).
Main Methods:
- Synthesis of an 18-mer amino-derivitized MORF dimerized using disuccinimidyl suberate (DSS).
- Purification of bivalent MORF via ion exchange chromatography.
- SPR analysis of bivalent and monovalent MORF binding to immobilized complementary biotinylated cDNA with varying spacing.
Main Results:
- Bivalent MORF was successfully synthesized with a yield of 45% and confirmed by MALDI-TOF mass spectroscopy.
- SPR data indicated bimolecular binding for bivalent MORF, evidenced by a significantly lower dissociation rate constant (10-fold decrease) and higher equilibrium constant (20-fold increase) compared to monovalent MORF.
- Association rate constants were similar between monovalent and bivalent MORFs, suggesting enhanced affinity is primarily due to reduced dissociation.
Conclusions:
- A readily synthesized bivalent MORF exhibits enhanced hybridization affinity compared to monovalent MORF.
- The observed lower dissociation and higher equilibrium constants confirm bimolecular binding of bivalent MORF.
- Bivalency in MORFs presents a promising strategy for improved pretargeting applications in molecular medicine.