Specific Loop Sequences Research Articles

Abstract 3090: Molecular structure of the major G-quadruplex formed in the PDGFR-β promoter nuclease hypersensitivity element and its binding with small molecules

Abstract Overexpression of platelet-derived growth factor receptor β (PDGFR-β) is associated with multiple cancers, making PDGFR-β an attractive target for anticancer drugs. Few strategies other than molecular targeting of the PDGFR-β protein or its cognate ligand have been reported for developing inhibitors for the PDGFR-β signaling pathway. DNA G-quadruplexes (G4s) formed in the GC-rich nuclease hypersensitivity element of the human PDGFR-β gene promoter have been found to inhibit PDGFR-β transcriptional activity, and stabilization of these G4s by small-molecule compounds could serve as a mechanism for cancer therapeutics. We have shown that the major G4 formed in the PDGFR-β promoter is from the central four G-runs, adopting an intramolecular parallel-stranded structure with a broken G-strand and contains three 1-nucleotide (nt) chain-reversal loops and one lateral loop. The novel folding of the PDGFR-β G4 highlights the inherent stability of the 1-nt loops in parallel-stranded G4 structures. Elucidating the structure of the major PDGFR-β promoter G4 is important for designing small-molecule drugs to specifically target this structure to inhibit gene transcription. Using nuclear magnetic resonance (NMR) spectroscopy, we have determined the potassium solution structure of this major G4 formed in the PDGFR-β promoter. Our structure showed a unique 3’ capping structure involving three guanine nucleotides in the lateral loop. This novel capping structure is determined by the specific loop sequence as well as the broken-stranded PDGFR-β G4 folding pattern. The unique 3’ capping structure could be specifically recognized by proteins and small molecule ligands. Unrestrained molecular dynamic calculations showed that this major PDGFR-β G4 and its 3’ capping structure are stable in aqueous environment on the ns time-scale. We also investigated the binding of small-molecules to the PDGFR-β G4 using a combination of NMR, circular dichroism (CD), and fluorescence based methods to identify compounds that can selectively bind the PDGFR-β G4 over other classic parallel-stranded G-quadruplexes, such as the c-MYC promoter G4. Our study demonstrates the structural diversity in promoter G4s which may enable specific recognition and the modulation of gene expression by small-molecule drugs. Citation Format: Clement Lin, Prashansa Agrawal, Yuwei Chen, Salil Kalarn, Nanjie Deng, Laurence Hurley, Danzhou Yang. Molecular structure of the major G-quadruplex formed in the PDGFR-β promoter nuclease hypersensitivity element and its binding with small molecules. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3090.

Expression and characterization of EF-hand I loop mutants of aequorin replaced with other loop sequences of Ca2+-binding proteins: an approach to studying the EF-hand motif of proteins.

The binding properties of Ca(2+) to EF-hand I of aequorin (AQ) were characterized by replacing the loop sequence of EF-hand I (AQ[I]) with other known loop sequences of Ca(2+)-binding proteins, including photoproteins (aequorin, clytin-I, clytin-II and mitrocomin), Renilla luciferin-binding protein (RLBP) and calmodulin (CaM). For evaluation of the binding affinity of Ca(2+) to AQ[I] mutants, the half-decay time of the maximum intensity in the luminescence reaction triggered by Ca(2+) was used as an indicator and 22 kinds of AQ[I] mutants were expressed in Escherichia coli cells. AQ[I] mutants replaced with the EF-hand I and EF-hand III from photoproteins showed sufficient luminescence activity, but it was not shown by other EF-hands from RLBP and CaM. An AQ[I] mutant with a lysine or arginine residue at the second position of the non-conserved amino acid residue showed a slow-decay pattern of luminescence, indicating that the Ca(2+)-binding affinity to aequorin was reduced by a positive charge at the second position of the loop sequence. The specific loop sequence of the EF-hand I motif in aequorin caused the specific Ca(2+)-triggered luminescence pattern.

Reversible Folding of Hyperstable RNA Tetraloops Using Molecular Dynamics Simulations

Structured RNAs exhibit a distinct preference for loops of precisely 4 nucleotides. Approximately 70% of these “tetraloops” are comprised of just three specific loop sequences: UUCG, GCAA, or CUUG. The abundance of these sequences is thermodynamic in origin, as each motif forms a unique network of non-canonical interactions within their loops that stabilize the folded state. Modification to the Amber force field enables the de-novo folding of three hyperstable RNA tetraloops to 1-3 A RMSD from their experimentally determined structures using molecular dynamics simulations initialized in the unfolded state. To study the thermodynamics and kinetics of folding on an RNA tetraloop we simulated the (rGCAA) tetraloop with stem lengths of two (octamer), and four (dodecamer) C-G base pairs. The thermodynamics is obtained from replica exchange molecular dynamics (REMD) simulations with overall sampling exceeding 300 microseconds. The kinetics of the octamer was studied in a 100 microseconds molecular dynamics simulation using the Anton supercomputer. The thermodynamics reveal that the octamer folds and unfolds reversibly. However, the dodecamer behaves glassy and adopts multiple metastable loop configurations that do not unfold in extensive REMD simulations. The ability to recapitulate the signature non-canonical interactions of the three most abundant hyperstable stem-loop motifs represents a significant step towards the accurate description of nucleic acid tertiary structures, dynamics and stability using unbiased all-atom molecular dynamics simulations.

ShRNAPred (version 1.0): An open source and standalone software for short hairpin RNA (shRNA) prediction.

The small hairpin RNAs (shRNA) are useful in many ways like identification of trait specific molecular markers, gene silencing and characterization of a species. In public domain, hardly there exists any standalone software for shRNA prediction. Hence, a software shRNAPred (1.0) is proposed here to offer a user-friendly Command-line User Interface (CUI) to predict ‘shRNA-like’ regions from a large set of nucleotide sequences. The software is developed using PERL Version 5.12.5 taking into account the parameters such as stem and loop length combinations, specific loop sequence, GC content, melting temperature, position specific nucleotides, low complexity filter, etc. Each of the parameters is assigned with a specific score and based on which the software ranks the predicted shRNAs. The high scored shRNAs obtained from the software are depicted as potential shRNAs and provided to the user in the form of a text file. The proposed software also allows the user to customize certain parameters while predicting specific shRNAs of his interest. The shRNAPred (1.0) is open access software available for academic users. It can be downloaded freely along with user manual, example dataset and output for easy understanding and implementation.AvailabilityThe database is available for free at http://bioinformatics.iasri.res.in/EDA/downloads/shRNAPred_v1.0.exe

Accurate curve fitting procedure for UV melting analysis of highly thermostable RNA hairpins

Thermostable RNA hairpins with specific loop sequences are critically important structural elements for proper folding of functional RNAs. Thermodynamic parameters of the structural elements are indispensable for secondary structure prediction. However when RNA folds into highly thermostable hairpin-loop structures, it is difficult to determine the melting temperature and other thermodynamic parameters from the normal UV melting analysis. In this study, we demonstrate a procedure to solve the problem using our software, Jfit. UV absorbance in the higher temperature region above 95 degrees C and the upper baseline were estimated using this analytical procedure. The ideal lines of -RlnK vs. 1/T were generated from the predicted thermodynamic parameters and the solution candidates were compared with the van't Hoff plot generated from the measured melting curve to judge their self-consistency. As a result, our analysis was able to reduce the number of the possible solution and to specify the best set of thermodynamic parameters for the stable hairpin formation.

Unique recognition style of tRNALeu by Haloferax volcaniiLeucyl-tRNA synthetase

The recognition manner of tRNALeu, a class II tRNA characterized by a long variable arm, by leucyl-tRNA synthetase from an extreme halophilic archaea, Haloferax volcanii, was studied using the in vitro transcription system. It was found that the discriminator base (A73) and the long variable arm, especially the specific loop sequence A47CG47D and U47H at the base of this helix, are significant for recognition by LeuRS. An appropriate stem length of the variable arm was also required. Base substitutions in the anticodon arm did not affect the leucylation activity. Transplantation of both the discriminator base and the variable arm of tRNALeu was not sufficient to introduce leucylation activity to tRNASer. Insertion of an additional nucleotide into the D-loop, which is not involved in the direct interaction with LeuRS, converted tRNASer to an efficient leucine acceptor. This suggests that differences in the tertiary structure play a key role in eliminating tRNASer. The sequence-specific recognition of the long variable arm of tRNALeu has not been observed in any of other organisms reported, such as Escherichia coli, yeast or human. On the other hand, the mode of discrimination from non-cognate tRNAs is similar to that in E. coli in that differences in the tertiary structure play a key role. Similarity extends to the substrate stringency, exemplified by a cross-species aminoacylation study showing that no class II tRNAs from E. coli or yeast can be leucylated by H. volcanii LeuRS. Our results have implications for the understanding of the evolution of the recognition system of class II tRNA.

Requirements for kissing-loop-mediated dimerization of human immunodeficiency virus RNA.

Sequences from the 5' end of type 1 human immunodeficiency virus RNA dimerize spontaneously in vitro in a reaction thought to mimic the initial step of genomic dimerization in vivo. Dimer initiation has been proposed to occur through a "kissing-loop" interaction involving a specific RNA stem-loop element designated SL1: the RNA strands first interact by base pairing through a six-base GC-rich palindrome in the loop of SL1, whose stems then isomerize to form a longer interstrand duplex. We now report a mutational analysis aimed at defining the features of SL1 RNA sequence and secondary structure required for in vitro dimer formation. Our results confirm that mutations which destroy complementarity in the SL1 loop abolish homodimer formation, but that certain complementary loop mutants can heterodimerize. However, complementarity was not sufficient to ensure dimerization, even between GC-rich loops, implying that specific loop sequences may be needed to maintain a conformation that is competent for initial dimer contact; the central GC pair of the loop palindrome appeared critical in this regard, as did two or three A residues which normally flank the palindrome. Neither the four-base bulge normally found in the SL1 stem nor the specific sequence of the stem itself was essential for the interaction; however, the stem structure was required, because interstrand complementarity alone did not support dimer formation. Electron microscopic analysis indicated that the RNA dimers formed in vitro morphologically resembled those isolated previously from retroviral particles. These results fully support the kissing-loop model and may provide a framework for systematically manipulating genomic dimerization in type 1 human immunodeficiency virus virions.

RNA Structural Requirements for RNA Binding, Replication, and Packaging in the Yeast Double-Stranded RNA Virus

Several sites of interaction with viral proteins have been mapped in the plus strand of the Saccharomyces cerevisiae double-stranded RNA virus, ScV. These include a site necessary and sufficient for viral particle binding to plus strands (VBS) and a site necessary and sufficient for interference with replication of viral plus strands (INS). We show that the INS and VBS are identical and that they are necessary and sufficient for packaging. One of the viral RNAs has two adjacent VBS, which have additive INS activity. The second VBS has similar affinity for viral particles as the first but its complex with particles exhibits faster dissociation. Binding to the two sites in the viral RNA is independent but equivalent. This may mean two recognition sites per particle. This is what would be expected for two molecules of the protein thought to be responsible for sequence specific recognition, the cap-pol fusion polypeptide, per particle. Comparison of the secondary structures of several binding sites, as determined by site-directed mutagenesis and by ribonuclease mapping of single- and double-stranded regions, reveals a requirement for a specific loop sequence and two stem regions, in which there is no sequence specificity. Contrary to what was described in previous work, a bulge loop, but not a "bulged" A residue is necessary for binding of the second VBS. A minimal region of 30 bases is enough to cause both packaging and replication.