Guanosine-rich sequences are prone to fold into four-stranded nucleic acid structures. Such quadruplex sequences have long been suspected to play important roles in regulatory processes within cells. Although DNA quadruplexes have been studied in great detail, four-stranded structures made up from RNA have received only minor attention, although it is known that RNA is able to form stable quadruplexes as well. Here, we compare quadruplex structures and stabilities of a variety of DNA and RNA sequences. We focus on well established DNA sequences and determine the topologies and stabilities of the corresponding RNA sequences by CD spectroscopy and CD thermal melting experiments. We find that the RNA sequences exclusively fold into the all-parallel conformation in contrast to the diverse topologies adopted by DNA quadruplexes. The thermal stabilities of the RNA structures rival those of the corresponding DNA sequences, often displaying enhanced stabilities compared to their DNA counterparts. Especially thermodynamically less stable sequences show a strong preference for potassium, with the RNA quadruplexes exhibiting much higher stabilities than the corresponding DNAs. The latter finding suggests that quadruplexes formed at critical positions in mRNAs might perturb gene expression to a larger extend than previously anticipated.

Aptamers are nucleic acids generated by in vitro selection techniques that are able to specifically bind to ligands such as proteins. 1 A series of aptamers targeting blood clotting factors have been described that are highly effective in inhibiting blood coagulation and thus act as anticoagulants. 2 In applications, such as cardiopulmonary bypass surgery, it is crucial to reverse the anticoagulant effect. For this reason, anticoagulant/antidote effector pairs are required, and only one such pair, consisting of heparin as anticoagulant and protamine as antidote, is currently used in daily clinical practice. However, the use of heparin is associated with potential drawbacks including immune reactions. 3 Aptamers that interfere with the blood clotting cascade in turn offer the advantage that antisense molecules can be easily designed that revert the aptamer's action, acting as antidote. 3 Recently, this approach has been developed further by introducing caged thrombin aptamers that can be controlled using light excitation. 4 The distinct structural features of the thrombin-inhibiting aptamer containing a G-quadruplex open an alternative approach to finetune aptamer function. Quadruplex-binding compounds such as the cationic porphyrin TMPyP4 (meso-5,10,15,20-tetrakis-(N-methyl-4-pyridyl)porphine) have attracted much attention since they bear the potential to interact with G-quadruplexes found in telomere ends of the chromosomes and promoters of proto-oncogenes, thereby often displaying anti-cancer activities. 5 Here, we have investigated if G-quadruplex-binding porphyrins are suited to control the anticoagulant activity of a G-quadruplex-containing aptamer that binds and inhibits human R-thrombin. We demonstrate that a cationic porphyrin, namely, TMPyP4, is able to bind to the aptamer and hence interferes with its active conformation. Consequently, TMPyP4 antagonizes the aptamer-mediated inhibition of blood clotting in a concentration-dependent manner, thus representing a true small molecule antidote of the anticoagulant DNA aptamer.Interactions of porphyrins with G-quadruplex structures are well characterized. 6 Cationic porphyrins such as TMPyP4 are known to bind to quadruplex structures such as the vertebrate telomeric sequence as well as G-rich promoter elements in proto-oncogenes such as c-myc. 7 Recently, we have engineered hammerhead ribozymes that are regulated by a TMPyP4-quadruplex interaction. 8 Nevertheless, so far, it has not been demonstrated that such interactions could be used to control the activity of functional aptamers containing G-quadruplex elements. In Figure 1A, the crystal structure of human R-thrombin in complex with the quadruplex-forming DNA aptamer inhibitor is shown. 9 Since the aptamer folds into an active G-quadruplex structure and thus allows specific binding to the blood clotting factor thrombin, interaction of G-quadruplex-binding porphyrins might interfere with the capability of the aptamer to bind to thrombin. In order to test this hypothesis, we have first characterized the in...

RNA interference is triggered by small hairpin precursors that are processed by the endonuclease dicer to yield active species such as siRNAs and miRNAs. To regulate the RNAi-mediated suppression of gene expression, we imagined a strategy that relies on the sequence-specific inhibition of shRNA precursor processing by immediate RNA-small molecule interactions. Here, we present a first step in this direction by augmenting shRNAs with guanosine-rich sequences that are prone to fold into four-stranded structures. The addition of small molecules that selectively bind to such quadruplex sequences should allow for the specific inhibition of dicing of shRNAs that contain suitable G-rich elements. In an attempt to find compounds that protect against dicer processing, we have examined the effects of quadruplex-binding compounds on the dicer processing of shRNAs containing G-quadruplexes. Although a variety of small molecules that are known to bind to quadruplexes inhibited in vitro dicing of shRNAs, only two substance classes, namely certain porphyrazines and bisquinolinium compounds, showed selective inhibition of G-rich shRNAs compared to control sequences lacking guanine-rich elements. The G-rich shRNAs displayed a potent knockdown of gene expression in mammalian cell culture, but the effect was not influenced by addition of the respective quadruplex-binding compounds.