Hairpins participating in folding of human telomeric sequence quadruplexes studied by standard and T-REMD simulations

DNA G-hairpins are potential key structures participating in folding of human telomeric guanine quadruplexes (GQ). We examined their properties by standard MD simulations starting from the folded state and long T-REMD starting from the unfolded state, accumulating ~130 \u3bcs of atomistic simulations. Antiparallel G-hairpins should spontaneously form in all stages of the folding to support lateral and diagonal loops, with sub-\u3bcs scale rearrangements between them. We found no clear predisposition for direct folding into specific GQ topologies with specific syn/anti patterns. Our key prediction stemming from the T-REMD is that an ideal unfolded ensemble of the full GQ sequence populates all 4096 syn/anti combinations of its four G-stretches. The simulations can propose idealized folding pathways but we explain that such few-state pathways may be misleading. In the context of the available experimental data, the simulations strongly suggest that the GQ folding could be best understood by the kinetic partitioning mechanism with a set of deep competing minima on the folding landscape, with only a small fraction of molecules directly folding to the native fold. The landscape should further include nonspecific collapse processes where the molecules move via diffusion and consecutive random rare transitions, which could, e.g., structure the propeller loops

Modulating RNA structure and catalysis: lessons from small cleaving ribozymes

RNA is a key molecule in life, and comprehending its structure/function relationships is a crucial step towards a more complete understanding of molecular biology. Even though most of the information required for their correct folding is contained in their primary sequences, we are as yet unable to accurately predict both the folding pathways and active tertiary structures of RNA species. Ribozymes are interesting molecules to study when addressing these questions because any modifications in their structures are often reflected in their catalytic properties. The recent progress in the study of the structures, the folding pathways and the modulation of the small ribozymes derived from natural, self-cleaving, RNA motifs have significantly contributed to today’s knowledge in the field

Base-flipping dynamics in a DNA hairpin processing reaction

Many enzymes that repair or modify bases in double-stranded DNA gain access to their substrates by base flipping. Although crystal structures provide stunning snap shots, biochemical approaches addressing the dynamics have proven difficult, particularly in complicated multi-step reactions. Here, we use protein–DNA crosslinking and potassium permanganate reactivity to explore the base-flipping step in Tn5 transposition. We present a model to suggest that base flipping is driven by a combination of factors including DNA bending and the intrusion of a probe residue. The forces are postulated to act early in the reaction to create a state of tension, relieved by base flipping after cleavage of the first strand of DNA at the transposon end. Elimination of the probe residue retards the kinetics of nicking and reduces base flipping by 50%. Unexpectedly, the probe residue is even more important during the hairpin resolution step. Overall, base flipping is pivotal to the hairpin processing reaction because it performs two opposite but closely related functions. On one hand it disrupts the double helix, providing the necessary strand separation and steric freedom. While on the other, transposase appears to position the second DNA strand in the active site for cleavage using the flipped base as a handle

Aptamers in oncology: a diagnostic perspective

Nucleic acid sequences can produce a wide variety of three-dimensional conformations. Some of these structural forms are able to interact with proteins and small molecules with high affinity and specificity. These sequences, comprising either double or single stranded oligonucleotides, are called 'aptamers' based on the Greek word aptus, which means 'to fit'. Using an efficient selection process, randomised oligonucleotide libraries can be rapidly screened for aptamers with the appropriate binding characteristics. This technology has spawned the development of a new class of oligonucleotide therapeutic products. However, while interest among pharmaceutical companies continues to grow with some candidates already in clinical trials and one in the market, there appears to be some reluctance to fully explore the diagnostic potential of this technology. This article will review aptamer developments in diagnostics, compare them with other oligonucleotide therapeutics and highlight both potentials and pitfalls of technological development in this area

Ratcheting synthesis

Synthetic chemistry has traditionally relied on reactions between reactants of high chemical potential and transformations that proceed energetically downhill to either a global or local minimum (thermodynamic or kinetic control). Catalysts can be used to manipulate kinetic control, lowering activation energies to influence reaction outcomes. However, such chemistry is still constrained by the shape of one-dimensional reaction coordinates. Coupling synthesis to an orthogonal energy input can allow ratcheting of chemical reaction outcomes, reminiscent of the ways that molecular machines ratchet random thermal motion to bias conformational dynamics. This fundamentally distinct approach to synthesis allows multi-dimensional potential energy surfaces to be navigated, enabling reaction outcomes that cannot be achieved under conventional kinetic or thermodynamic control. In this Review, we discuss how ratcheted synthesis is ubiquitous throughout biology and consider how chemists might harness ratchet mechanisms to accelerate catalysis, drive chemical reactions uphill and programme complex reaction sequences.<br/

Facilitation of a structural transition in the polypurine/polypyrimidine tract within the proximal promoter region of the human VEGF gene by the presence of potassium and G-quadruplex-interactive agents

The proximal promoter region of the human vascular endothelial growth factor (VEGF) gene contains a polypurine/polypyrimidine tract that serves as a multiple binding site for Sp1 and Egr-1 transcription factors. This tract contains a guanine-rich sequence consisting of four runs of three or more contiguous guanines separated by one or more bases, corresponding to a general motif for the formation of an intramolecular G-quadruplex. In this study, we observed the progressive unwinding of the oligomer duplex DNA containing this region into single-stranded forms in the presence of KCl and the G-quadruplex-interactive agents TMPyP4 and telomestatin, suggesting the dynamic nature of this tract under conditions which favor the formation of the G-quadruplex structures. Subsequent footprinting studies with DNase I and S1 nucleases using a supercoiled plasmid DNA containing the human VEGF promoter region also revealed a long protected region, including the guanine-rich sequences, in the presence of KCl and telomestatin. Significantly, a striking hypersensitivity to both nucleases was observed at the 3′-side residue of the predicted G-quadruplex-forming region in the presence of KCl and telomestatin, indicating altered conformation of the human VEGF proximal promoter region surrounding the guanine-rich sequence. In contrast, when specific point mutations were introduced into specific guanine residues within the G-quadruplex-forming region (Sp1 binding sites) to abolish G-quadruplex-forming ability, the reactivity of both nucleases toward the mutated human VEGF proximal promoter region was almost identical, even in the presence of telomestatin with KCl. This comparison of wild-type and mutant sequences strongly suggests that the formation of highly organized secondary structures such as G-quadruplexes within the G-rich region of the human VEGF promoter region is responsible for observed changes in the reactivity of both nucleases within the polypurine/polypyrimidine tract of the human VEGF gene. The formation of the G-quadruplex structures from this G-rich sequence in the human VEGF promoter is further confirmed by the CD experiments. Collectively, our results provide strong evidence that specific G-quadruplex structures can naturally be formed by the G-rich sequence within the polypurine/polypyrimidine tract of the human VEGF promoter region, raising the possibility that the transcriptional control of the VEGF gene can be modulated by G-quadruplex-interactive agents

Quantitative design and experimental validation for a single-molecule DNA nanodevice transformable among three structural states

In this work, we report the development and experimental validation of a coupled statistical thermodynamic model allowing prediction of the structural transitions executed by a novel DNA nanodevice, for quantitative operational design. The efficiency of target structure formation by this nanodevice, implemented with a bistable DNA molecule designed to transform between three distinct structures, is modeled by coupling the isolated equilibrium models for the individual structures. A peculiar behavior is predicted for this nanodevice, which forms the target structure within a limited temperature range by sensing thermal variations. The predicted thermal response is then validated via fluorescence measurements to quantitatively assess whether the nanodevice performs as designed. Agreement between predictions and experiment was substantial, with a 0.95 correlation for overall curve shape over a wide temperature range, from 30C to 90C. The obtained accuracy, which is comparable to that of conventional melting behavior prediction for DNA duplexes in isolation, ensures the applicability of the coupled model for illustrating general DNA reaction systems involving competitive duplex formation. Finally, tuning of the nanodevice using the current model towards design of a thermal band pass filter to control chemical circuits, as a novel function of DNA nanodevices is proposed

Study of complex RNA function modulated by small molecules: the development of RNA directed small molecule library and probing the S-adenosyl methionine discrimination between on and off conformational states of the SAM-I riboswitch

RNA recently remained unexploited and is now drawing interest as a potential drug target. The methodology and available drug libraries for RNA targeting/screening are in rudimentary stages. The interactions made by ligands with RNA can be explored for RNA based drug development. The dissertation is composed of 4 chapters. The first chapter focuses on the structural features of RNA and the attempts made to target RNA previously. The second chapter focuses on the development of a small molecule library enriched with substructures derived from RNA binding ligands. For this study a fragment-based approach (fragment based approach is detailed in chapter 2) is used in order to accommodate the conformational flexibility of RNA. The library molecules are used for screening against suitable RNA targets using NMR. We identified at least 5 ligands out of which 2 are novel ligands binding to the ribosomal 16s rRNA. The third chapter is focused on the role of small molecules in inducing conformational changes in an RNA genetic regulatory element called the S-Adenosyl methionine (SAM) SAM-I riboswitch. The mechanistic features of the SAM-I riboswitch to understand the basis for specificity and discrimination and its gene regulation mechanism are reported. To address the conformational dynamics Bacillus subtilis and Thermoanearobacter tencongenesis SAM-I riboswitches in response to SAM binding several conformer mimics are designed, synthesized and characterized using NMR, equilibrium dialysis, and inline probing. The study shows that apart from the conserved residues of the binding pocket, residues downstream of the binding pocket are involved in detecting SAM and assist the binding of SAM to the riboswitch with weak affinity. Our data highlights the capacity of a so-called antiterminator helix from the expression platform to assist the formation of a partial P1 helix of the aptamer domain. A stable P1 is involved in recognition and tight binding of SAM. Our in vitro experiments suggest that the riboswitch could switch from an unbound conformation to tightly SAM bound structure through weakly binding intermediate structures in the presence of the small molecule SAM. The future directions are included in the fourth chapter along with the conclusions