PAPER www.rsc.org/obc | Organic & Biomolecular Chemistry Cooperative binding and self-assembling behavior of cationic low molecular-weight dendrons with RNA molecules†‡ Jiehua Zhou, a Jiangyu Wu, a, f Xiaoxuan Liu, a Fanqi Qu, a Mu Xiao, b Yi Zhang, b Laurence Charles, c Cheng-Cai Zhang d ,e and Ling Peng* a, f Received 3rd November 2005, Accepted 2nd December 2005 First published as an Advance Article on the web 9th January 2006 DOI: 10.1039/b515667j Tri(ethylene glycol) derived, low molecular-weight dendrons with various amine end groups were synthesized and characterized for their properties of binding and self-assembling with RNA using the Candida ribozyme as a model RNA molecule. These dendritic compounds form stable complexes and well-defined nanoscale particles with RNA molecules via electrostatic interactions and self-assembly process, while leaving the other terminal of the tri(ethylene glycol) chain accessible for targeting. This suggests that dendrimers of this type hold great promise for specific RNA targeting and RNA delivery. Introduction Dendrimers are perfectly structured molecules with large numbers of cascade-branched units emanating from a focal point, resulting in densely packed end groups at the molecular surface. 1 This feature of dendrimers has been widely used to generate multivalent interactions and cooperative effects in order to amplify weak interactions and obtain special functions and properties. 1–4 One of the main biological applications of dendrimers is dendrimer- based gene transfer, which involves polycationic dendrimers such as polyamidoamine (PAMAM), 5 polylysine 6 and poly(propylene imine) (PPI). 7 Under physiological conditions these dendrimers have positively charged amine end groups at the dendrimer surface. They self-assemble with DNA via electrostatic interactions, which result in cooperative binding and dense packing between the dendrimers and DNA molecules. In general, DNA binding and delivery are more effective with dendrimers of higher-generation or structurally fractured systems. 5,8 Low molecular-mass dendrons were recently reported to have a high binding affinity with DNA and to be capable of efficient gene delivery. 7,9 Polycationic dendrons may be used in a similar way with RNA, another class of biologically relevant nucleic acids which are involved in a wide range of important biological functions such as protein synthesis, post-transcriptional RNA processing, regulation of a College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, 430072, P. R. China b College of Life Sciences, Wuhan University, Wuhan, 430072, P. R. China c JE 2421 TRACES, Facult´ e des Sciences et Techniques de St. J´ erˆ ome, Avenue Escadrille Normandie-Niemen, 13397, Marseille Cedex 13, France d Laboratoire de Chimie Bact´ erienne, CNRS UPR 9043, 31 Chemin Joseph Aiguier, 13402, Marseille cedex 20, France e National Key Laboratory of Agricultural Microbiology, Huazhong Agricul- tural University, Wuhan, 430070, P. R. China f D´ epartement de Chimie, CNRS UMR 6114, 163, Avenue de Luminy, 13288, Marseille, France. E-mail: ling.peng@univmed.fr; Fax: +33 4 91 82 93 01; Tel: +33 4 91 82 91 54 †Electronic supplementary information (ESI) available: Detailed experi- mental procedures for dendrimer synthesis and characterization as well as studies on RNA/dendrimer complexes using different methods. See DOI: 10.1039/b515667j ‡ Zhou JH and Wu JY contributed equally to this work. gene expression and retroviral replication, and are emerging as both important drug targets and versatile therapeutic agents. 10 However, far fewer efforts have been made so far with RNA. 11 We recently established that polycationic PAMAM dendrimers interact strongly with RNA ribozymes via cooperative electrostatic interactions, thus strongly inhibiting the catalytic activities of ribozymes. 11 This finding opens up new perspectives for using polycationic PAMAM dendrimers for both RNA targeting and RNA delivery purposes. 12 The possibility of closely controlling the size, shape and surface chemistry of dendrimers 1 gives us an opportunity of creating a repertoire of structure-, size- and shape-tailored dendrimers binding to various RNA molecules as required. In our ongoing project focusing on RNA targeting and RNA delivery, we are interested in developing low molecular-mass dendrons with well- defined structures that can bind and assemble with RNA molecules via cooperative electrostatic interactions, as well as being able to conjugate a ligand for eventual specific targeting purposes. Systems of this kind can help to throw light on structure–activity relationships and may have better chances of being used for cell- or organ-specific delivery of nucleic acids. 13 Tri(ethylene glycol) is a low molecular-weight compound of poly(ethylene glycol)s, known to be highly water-soluble, non-immunogenic, and biocompatible. 14 Tri(ethylene glycol) is widely used as a linker to connect two distinct functionalities in bioconjugated molecules. By promoting dendrimer growth at only one end of tri(ethylene glycol), we can obtain fan- shaped PAMAM dendrimers with various amine end groups for RNA binding (Scheme 1), while the other terminal of the tri(ethylene glycol) chain can be coupled with a specific ligand for targeting purposes. The tri(ethylene glycol) part is neutral and so will not create electrostatic interactions and may also have steric hindrance effects on RNA binding. It is therefore necessary to demonstrate whether these tri(ethylene glycol) derived PAMAM dendrons still hold strong RNA binding properties. Here we report on the synthesis of tri(ethylene glycol) derived PAMAM dendritic constructs and on studies in which we assessed their binding properties and self-assembling behavior with RNA molecules. This journal is © The Royal Society of Chemistry 2006 Org. Biomol. Chem., 2006, 4, 581–585 | 581