G-quadruplexes are well-characterized, four-stranded conformations comprised of stacked arrangements of guanine nucleobase tetrads, stabilized by Hoogsteen hydrogen bonding and centrally coordinated cations. The stable formation of quadruplexes has been studied for both DNA and RNA sequences. Many such sequences were characterized in the promoter regions of proto-oncogenes like c-MYC, c-MYB, BCL2, KRAS and cKIT, often coinciding with or in near vicinity to a transcription factor binding site, and hence potentially acting as cis-regulatory elements of transcription. Moreover, they have been reported to exclude nucleosome occupied regions, which could also have regulatory significance. Together, these studies encouraged the design and synthesis of quadruplex-specific small molecules for telomerase inhibition and regulation of transcription. Indeed, various small molecules have been shown to interact and stabilize DNA quadruplexes, in vitro, and are thus suspected to interfere with DNA-related processes, in vivo. In particular, interference of quadruplex binding molecules with telomeric functions and transcription of oncogenes has been investigated. However, it is likely that the probability of formation, folding kinetics, life-time, and thermodynamic stability of quadruplex structures are highly dependent on the local primary sequence (such as number and length of guanosine runs as well as loop lengths and compositions) but also on the nucleic acid backbone (DNA vs. RNA) and the native status of the nucleic acid (single-stranded vs. double-stranded). There is currently a large body of in vitro data concerning the influence of these structural features on quadruplex formation and stability. For instance, the formation of a DNA quadruplex in the presence of the complementary strand is expected to be transient due to the thermodynamically favored duplex state. Hence, DNA quadruplexes are expected to occur with a higher probability during processes that require duplex strand separation, such as at replication forks, transcription bubbles, and also at the telomeric single-stranded overhang. In contrast, due to their single-stranded appearance RNA sequences are prone to form thermodynamically stable higherorder structures, such as hairpins and quadruplexes. Although most studies are concerned with DNA quadruplex functions, the interest in four-stranded RNA sequences is increasing. For instance, human telomeres have been found to be transcribed into approximately 100–9000 nucleotide telomeric repeat-containing RNAs (termed TERRA) and are predicted to form RNA quadruplexes, in vivo; this suggests a role for these molecules in telomere regulation and maintenance. Moreover, recent studies have employed computational searches for G-quadruplex forming sequences in human mRNAs and found a higher predominance in 5’-UTRs than statistically expected, indicating a possible regulatory function for these motifs. We have presented strong evidence for RNA quadruplex formation in Escherichia coli by showing that translation initiation gets inhibited if folding interferes with the accessibility of the ribosome binding site. Similar inhibitory effects were demonstrated for natural 5’-UTR-based RNA quadruplexes in mammalian cell culture for the expression of some genes. In addition, we introduced various symmetric RNA quadruplexes into the 5’-UTR of a reporter gene, and found that the level of suppression is proportional to the thermodynamic stability of the quadruplex motif. Since the loop length and number of G-rich stretches are important factors determining the stability of quadruplex motifs, we systematically varied these parameters in order to demonstrate that the extent of inhibition of gene expression correlated with the thermodynamic stability of the four-stranded motifs. When we investigated naturally occurring 5’-UTR quadruplexes from NRAS, CHST2, MAPK2 and PCGF2 genes in our setup, the suppressive effect of these sequences could be approximated by comparison to the symmetric sequences of similar architectures. Since the studied series of symmetric quadruplexes proved to produce very predictable and reliable effects, we chose these constructs in order to evaluate the possibility to further stabilize quadruplex formation by addition of quadruplex-binding compounds. Since most reported quadruplex binders stabilize the four-stranded fold (mostly determined for DNA quadruplexes), a further enhancement of the suppressive effects on gene expression could be expected upon application of such substances. We investigated three bisquinolinium compounds displaying either a pyridine dicarboxamide core, namely 360A (also called PDC for pyridine dicarboxamide) or a phenanthrolinedicarboxamide core, namely PhenDC3 and PhenDC6, two isomers differing by the orientation of the attached quinoline moiety (Figure 1A). This set of compounds has been selected on the basis of previous studies that have demonstrated their efficacy to bind and stabilize DNA quadruplexes, and their potential to perturb the expression of targeted DNA quadruplex-forming sequences. In addition, these compounds exhibit high selectivity for DNA quadruplexes compared to duplex DNA. This binding preference is attributed to the bent shape of the [a] Dr. K. Halder, M. Benzler, Prof. Dr. J. S. Hartig Department of Chemistry, University of Konstanz 78457 Konstanz (Germany) E-mail : joerg.hartig@uni-konstanz.de [b] E. Largy, Prof. Dr. M.-P. Teulade-Fichou Institut Curie Centre de Recherche, CNRS-UMR 176 Universit Paris XI, 91405 Orsay (France) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201100228.
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Aug 13, 2012
Nicole L Batenburg + 4
Open Access
