The present research represents a step towards understanding the mechanism by which biological macromolecules fold into their functional native conformations. The use of NMR spectroscopy and other methods enabled detection of pre-folded structures of DNA oligonucleotides derived from human telomeric repeat that exist in solution before completely formed G-quadruplexes upon addition of cations. Unexpectedly well-defined pre-folded structures composed of base triples for studied oligonucleotides were detected at certain pH and temperature. These kinds of structures were up to now only hypothesized as intermediates in the folding process. All determined pre-folded structures (with base pairs and base triples, with antiparallel chair-like topology as well as hairpin) showed that the first and the second guanine-tracts are connected in antiparallel orientation. This structural feature could be the main reason for different folding of DNA oligonucleotides derived from human telomeric repeat into their final G-quadruplex structures. Characterization of long-lived pre-folded structures is essential not only to establish mechanisms of G-quadruplex formation as they can represent on- or off-pathway intermediates, but can be used in development of novel type of selective ligands that will target their peculiar structural elements at pre-quadruplex states that are intrinsically more dynamic and can bind heterocyclic ligands through a privileged conformation.
COBISS.SI-ID: 6795290
In collaboration with colleagues from Italy, we investigated the formation of i-motives in long terminal repeats of the HIV-1 gene promoter. I-motifs are non-canonical nucleic acid structures characterized by intercalated H-bonds between hemiprotonated cytosines. The evidence for the involvement of i-motif structures in the regulation of cellular processes in human cells has steadily increased in recent years. However, i-motifs within non-human genomes have never been studied. Here we report on the characterization of i-motifs within the long terminal repeat (LTR) promoter of the HIV-1 provirus genome. Biophysical and biochemical analysis revealed the formation of a predominant i-motif with an unprecedented loop composition. By using nuclear magnetic resonance, we have shown the formation of Watson-Crick G-C base pairs in the long loop, which probably improves the overall structural stability. Pull-down experiments in combination with mass spectrometry and protein cross-linking analysis showed that the LTR i-motif is recognized by the cellular protein hnRNP K, which induces folding under physiological conditions. In addition, hnRNP K-silencing led to increased LTR promoter activity, confirming the protein's ability to stabilize the i-motif-forming sequence, which in turn regulates LTR -mediated HIV-1 transcription. These findings provide new insights into the complexity of the HIV-1 virus and lay the foundation for an innovative antiviral drug design based on the ability to selectively detect and target the HIV-1 LTR i motif.
COBISS.SI-ID: 40309253
Fluorinated RNA molecules, particularly 2'-F RNA, have found a wide range of applications in RNA therapeutics, RNA aptamers, and ribozymes and as 19F NMR probes for elucidating RNA structure. Owing to the instability of 4'-F ribonucleosides, synthesis of 4'-F-modified RNA has long been a challenge. In this study, we developed a strategy for synthesizing a 4'-F-uridine (4'FU) phosphoramidite, and we used it to prepare 4'-F RNA successfully. In the context of an RNA strand, 4'FU, which existed in a North conformation, was reasonably stable and resembled unmodified uridine well. The 19F NMR signal of 4'-FU was sensitive to RNA secondary structure, with a chemical shift dispersion as large as 4 ppm (compared with (1 ppm for 2'FU), which makes it a valuable probe for discriminating single-stranded RNA and A-type, B-type, and mismatched duplexes. In addition, we demonstrated that because RNA-processing enzymes treated 4'FU the same as unmodified uridine, 4'FU could be used to monitor RNA structural dynamics and enzyme-mediated RNA processing. Taken together, our results indicate that 4'-F RNA represents a probe with wide utility for elucidation of RNA structure and function by means of 19F NMR spectroscopy.
COBISS.SI-ID: 6801434