Nucleic Acid Conformation Research Articles

FRET-Filtered Spectroscopy to Simultaneously Probe Local and Global Conformations of Nucleic Acids

FRET-Filtered Spectroscopy to Simultaneously Probe Local and Global Conformations of Nucleic Acids

FTIR-based spectrum of salivary exosomes coupled with computational-aided discriminating analysis in the diagnosis of oral cancer.

To determine the Fourier-transform infrared (FTIR) spectra of salivary exosomes from oral cancer (OC) patients and healthy individuals (HI) and to assess its diagnostic potential using computational-aided models. Whole saliva samples were collected from 21 OC patients and 13 HI. Exosomes were pelleted using differential centrifugation (12,000g, 120,000g). The mid-infrared (IR) absorbance spectra (900-5000cm- 1 range) were measured using MIR8025 Oriel Fourier-transform IR equipped with a PIKE MIRacle ZnSe attenuated total reflectance attachment. Machine learning techniques, utilized to build discrimination models for the absorbance data of OC and HI, included the principal component analysis-linear discriminant analysis (PCA-LDA) and support vector machine (SVM) classification. Sensitivity, specificity and the area under the receiver operating characteristic curve were calculated. IR spectra of OC were consistently different from HI at 1072cm- 1 (nucleic acids), 2924cm- 1 and 2854cm- 1 (membranous lipids), and 1543cm- 1 (transmembrane proteins). The PCA-LDA discrimination model correctly classified the samples with a sensitivity of 100%, specificity of 89% and accuracy of 95%, and the SVM showed a training accuracy of 100% and a cross-validation accuracy of 89%. We showed the specific IR spectral signature for OC salivary exosomes, which was accurately differentiated from HI exosomes based on detecting subtle changes in the conformations of proteins, lipids and nucleic acids using optimized artificial neural networks with small data sets. This non-invasive method should be further investigated for diagnosis of oral cancer at its very early stages or in oral lesions with potential for malignant transformation.

Characterization of CD133+/CD44+ human prostate cancer stem cells with ATR-FTIR spectroscopy.

Current cancer treatments destroy the tumor mass but cannot prevent the recurrence of cancer. The heterogeneous structure of the tumor mass includes cancer stem cells that are responsible for tumor relapse, treatment resistance, invasion and metastasis. The biology of these cells is still not fully understood; therefore, effective treatments cannot be developed sufficiently. Herein, attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy, combined with unsupervised multivariate analysis, was applied to prostate cancer stem cells (CSCs), non-stem cancer cells (non-CSCs) and normal prostate epithelial cells to elucidate the molecular mechanisms and features of CSCs, which are crucial to improving the target specific therapies. This work revealed the spectral differences in the cellular mechanisms and biochemical structures among three different cell types. Particularly, prostate CSCs exhibit differences in the lipid composition and dynamics when compared to other cell types. CSCs also harbor pronounced differences in their major cellular macromolecules, including differences in the protein amount and content (mainly α-helices), the abundance of nucleic acids (DNA/RNA), altered nucleic acid conformation and carbohydrate composition. Interestingly, macromolecules containing the C[double bond, length as m-dash]O groups and negatively charged molecules having the COO- groups are abundant in prostate CSCs in comparison to prostate non-CSCs and normal prostate cells. Overall, this study demonstrates the potential use of ATR-FTIR spectroscopy as a powerful tool to obtain new insights into the understanding of the CSC features, which may provide new strategies for cancer treatment by selectively targeting the CSCs.

Soil Particles and Phenanthrene Interact in Defining the Metabolic Profile of Pseudomonas putida G7: A Vibrational Spectroscopy Approach.

In soil, organic matter and mineral particles (soil particles; SPs) strongly influence the bio-available fraction of organic pollutants, such as polycyclic aromatic hydrocarbons (PAHs), and the metabolic activity of bacteria. However, the effect of SPs as well as comparative approaches to discriminate the metabolic responses to PAHs from those to simple carbon sources are seldom considered in mineralization experiments, limiting our knowledge concerning the dynamics of contaminants in soil. In this study, the metabolic profile of a model PAH-degrading bacterium, Pseudomonas putida G7, grown in the absence and presence of different SPs (i.e., sand, clays and humic acids), using either phenanthrene or glucose as the sole carbon and energy source, was characterized using vibrational spectroscopy (i.e., FT-Raman and FT-IR spectroscopy) and multivariate classification analysis (i.e., PLS-DA). The different type of SPs specifically altered the metabolic profile of P. putida, especially in combination with phenanthrene. In comparison to the cells grown in the absence of SPs, sand induced no remarkable change in the metabolic profile of the cells, whereas clays and humic acids affected it the most, as revealed by the higher discriminative accuracy (R2, RMSEP and sensitivity) of the PLS-DA for those conditions. With respect to the carbon-source (phenanthrene vs. glucose), no effect on the metabolic profile was evident in the absence of SPs or in the presence of sand. On the other hand, with clays and humic acids, more pronounced spectral clusters between cells grown on glucose or on phenanthrene were evident, suggesting that these SPs modify the way cells access and metabolize PAHs. The macromolecular changes regarded mainly protein secondary structures (a shift from α-helices to β-sheets), amino acid levels, nucleic acid conformation and cell wall carbohydrates. Our results provide new interesting evidences that SPs specifically interact with PAHs in defining bacteria metabolic profiles and further emphasize the importance of studying the interaction of bacteria with their surrounding matrix to deeply understand PAHs degradation in soils.

Above and Beyond Watson and Crick: Guanine Quadruplex Structures and Microbes.

Advances in understanding mechanisms of nucleic acids have revolutionized molecular biology and medicine, but understanding of nontraditional nucleic acid conformations is less developed. The guanine quadruplex (G4) alternative DNA structure was first described in the 1960s, but the existence of G4 structures (G4-S) and their participation in myriads of biological functions are still underappreciated. Despite many tools to study G4s and many examples of roles for G4s in eukaryotic molecular processes and issues with uncontrolled G4-S formation, there is relatively little knowledge about the roles of G4-S in viral or prokaryotic systems. This review summarizes the state of the art with regard to G4-S in eukaryotes and their potential roles in human disease before discussing the evidence that G4-S have equivalent importance in affecting viral and bacterial life.

Polyamines protect nucleic acids against depurination.

Depurination is accelerated by heat and reactive oxygen species under physiological conditions. We previously reported that polyamines are involved in mitigation of heat shock and oxidative stresses through stimulation of the synthesis of heat shock and antioxidant proteins. This time, we investigated whether polyamines are directly involved in protecting nucleic acids from thermal depurination induced by high temperature. The suppressing efficiencies of depurination of DNA by spermine, caldopentamine and caldohexamine in the presence of 1 mM Mg2+, were approximately 50%, 60% and 80%, respectively. Mg2+ also protected nucleic acids against depurination but to a lesser degree than polyamines. Longer unusual polyamines were more effective at protecting DNA against depurination compared to standard polyamines. The tRNA depurination suppressing efficiencies of spermine, caldopentamine and caldohexamine in the presence of 1 mM Mg2+, were approximately 60%, 70% and 80%, respectively. Standard polyamines protected tRNA and ribosomes more effectively than DNA against thermal depurination. Branched polyamines such as mitsubishine and tetrakis(3-aminopropyl)ammonium also protected RNA more effectively than DNA against depurination. These results suggest that the suppressing effect of depurination of nucleic acids (DNA and RNA) depends on the types of polyamines: i.e. to maintain functional conformation of nucleic acids at high temperature, longer and branched polyamines play important roles in protecting nucleic acids from depurination compared to standard polyamines and Mg2+.

Bromide as a Robust Backfiller on Gold for Precise Control of DNA Conformation and High Stability of Spherical Nucleic Acids

Functionalization of a gold surface with DNA is often complicated by kinetic traps from unintended DNA base adsorption. Herein, we communicate that Br- serves as a robust backfilling agent displacing selected DNA bases on gold. Traditional thiol backfillers are too strong, while even 300 mM Br- is well tolerated. Conjugates prepared with Br- hybridize 10-fold faster and resist DNA release with better colloidal stability yielding highly sensitive probes. From colorimetric and Raman assays, adsorption affinity ranks as F- < T ≈ Cl- < C < G ≈ Br- < A < I-, allowing Br- to displace nonpoly-A sequences from gold. This well-controlled biointerface will impact biosensing, drug delivery, and directed assembly of nanomaterials.

Mechanism of polypurine tract primer generation by HIV-1 reverse transcriptase

HIV-1 reverse transcriptase (RT) possesses both DNA polymerase activity and RNase H activity that act in concert to convert single-stranded RNA of the viral genome to double-stranded DNA that is then integrated into the DNA of the infected cell. Reverse transcriptase–catalyzed reverse transcription critically relies on the proper generation of a polypurine tract (PPT) primer. However, the mechanism of PPT primer generation and the features of the PPT sequence that are critical for its recognition by HIV-1 RT remain unclear. Here, we used a chemical cross-linking method together with molecular dynamics simulations and single-molecule assays to study the mechanism of PPT primer generation. We found that the PPT was specifically and properly recognized within covalently tethered HIV-1 RT–nucleic acid complexes. These findings indicated that recognition of the PPT occurs within a stable catalytic complex after its formation. We found that this unique recognition is based on two complementary elements that rely on the PPT sequence: RNase H sequence preference and incompatibility of the poly(rA/dT) tract of the PPT with the nucleic acid conformation that is required for RNase H cleavage. The latter results from rigidity of the poly(rA/dT) tract and leads to base-pair slippage of this sequence upon deformation into a catalytically relevant geometry. In summary, our results reveal an unexpected mechanism of PPT primer generation based on specific dynamic properties of the poly(rA/dT) segment and help advance our understanding of the mechanisms in viral RNA reverse transcription.

Polyamines and Their Role in Virus Infection.

Polyamines are small, abundant, aliphatic molecules present in all mammalian cells. Within the context of the cell, they play a myriad of roles, from modulating nucleic acid conformation to promoting cellular proliferation and signaling. In addition, polyamines have emerged as important molecules in virus-host interactions. Many viruses have been shown to require polyamines for one or more aspects of their replication cycle, including DNA and RNA polymerization, nucleic acid packaging, and protein synthesis. Understanding the role of polyamines has become easier with the application of small-molecule inhibitors of polyamine synthesis and the use of interferon-induced regulators of polyamines. Here we review the diverse mechanisms in which viruses require polyamines and investigate blocking polyamine synthesis as a potential broad-spectrum antiviral approach.

Chemoselective Modification of Vinyl DNA by Triazolinediones.

A new method for the post-synthetic modification of nucleic acids was developed that involves mixing a phenyl triazolinedione (PTAD) derivative with DNA containing a vinyl nucleobase. The resulting reactions proceeded through step-wise mechanisms, giving either a formal [4+2] cycloaddition product, or, depending on the context of nucleobase, PTAD addition along with solvent trapping to give a secondary alcohol in water. Catalyst-free addition between PTAD and the terminal alkene of 5-vinyl-2'-deoxyuridine (VdU) was exceptionally fast, with a second-order rate constant of 2×103 m-1 s-1 . PTAD derivatives selectively reacted with VdU-containing oligonucleotides in a conformation-selective manner, with higher yields observed for G-quadruplex versus duplex DNA. These results demonstrate a new strategy for copper-free bioconjugation of DNA that can potentially be used to probe nucleic acid conformations in cells.

Chemoselective Modification of Vinyl DNA by Triazolinediones

AbstractA new method for the post‐synthetic modification of nucleic acids was developed that involves mixing a phenyl triazolinedione (PTAD) derivative with DNA containing a vinyl nucleobase. The resulting reactions proceeded through step‐wise mechanisms, giving either a formal [4+2] cycloaddition product, or, depending on the context of nucleobase, PTAD addition along with solvent trapping to give a secondary alcohol in water. Catalyst‐free addition between PTAD and the terminal alkene of 5‐vinyl‐2′‐deoxyuridine (VdU) was exceptionally fast, with a second‐order rate constant of 2×103 m−1 s−1. PTAD derivatives selectively reacted with VdU‐containing oligonucleotides in a conformation‐selective manner, with higher yields observed for G‐quadruplex versus duplex DNA. These results demonstrate a new strategy for copper‐free bioconjugation of DNA that can potentially be used to probe nucleic acid conformations in cells.

Impact of modified ribose sugars on nucleic acid conformation and function

Abstract The modification of the ribofuranose in nucleic acids is a widespread method of manipulating the activity of nucleic acids. These alterations, however, impact the local conformation and chemical reactivity of the sugar. Changes in the conformation and dynamics of the sugar moiety alter the local and potentially global structure and plasticity of nucleic acids, which in turn contributes to recognition, binding of ligands and enzymatic activity of proteins. This review article introduces the conformational properties of the (deoxy)ribofuranose ring and then explores sugar modifications and how they impact local and global structure and dynamics in nucleic acids.

The cellular protein hnRNP A2/B1 enhances HIV-1 transcription by unfolding LTR promoter G-quadruplexes

G-quadruplexes are four-stranded conformations of nucleic acids that act as cellular epigenetic regulators. A dynamic G-quadruplex forming region in the HIV-1 LTR promoter represses HIV-1 transcription when in the folded conformation. This activity is enhanced by nucleolin, which induces and stabilizes the HIV-1 LTR G-quadruplexes. In this work by a combined pull-down/mass spectrometry approach, we consistently found hnRNP A2/B1 as an additional LTR-G-quadruplex interacting protein. Surface plasmon resonance confirmed G-quadruplex specificity over linear sequences and fluorescence resonance energy transfer analysis indicated that hnRNP A2/B1 is able to efficiently unfold the LTR G-quadruplexes. Evaluation of the thermal stability of the LTR G-quadruplexes in different-length oligonucleotides showed that the protein is fit to be most active in the LTR full-length environment. When hnRNP A2/B1 was silenced in cells, LTR activity decreased, indicating that the protein acts as a HIV-1 transcription activator. Our data highlight a tightly regulated control of transcription based on G-quadruplex folding/unfolding, which depends on interacting cellular proteins. These findings provide a deeper understanding of the viral transcription mechanism and may pave the way to the development of drugs effective against the integrated HIV-1, present both in actively and latently infected cells.

Conformations of Single-Stranded Nucleic Acids in Solution

Conformations of Single-Stranded Nucleic Acids in Solution

Abstract 3909: Interactions of glioma cells with chlorotoxin: an ATR-FTIR spectroscopy study

Abstract Chlorotoxin is a scorpion-derived peptide that preferentially binds to tumor cells of neuroectodermal origin, like glioma, but not to normal non-transformed cells. We have previously reported the development of a targeted nanodelivery system using chlorotoxin. Chlorotoxin has been studied as an imaging and targeting agent for drug and radioisotope delivery, but the mechanism of interaction of chlorotoxin with cancer cells has not been well understood and it needs to be further evaluated in order to optimize its use as a targeting compound for cancer cells. In this study, we used U87 human glioma cell line to evaluate the binding kinetics of chlorotoxin by Attenuated Total Reflection Fourier Transform Infra-Red (ATR-FTIR) spectroscopy. As a sensitive and label free technique, ATR-FTIR is a powerful diagnostic tool for the comparison of cancer cells with normal cells. First, we characterized the signature spectra of chlorotoxin and U87 cells by assigning and evaluating the infrared absorption bands and their second derivatives. Next, we studied the spectral differences between U87 cells incubated with and without chlorotoxin for incremental time periods ranging from 15 minutes to 24 hours. We also examined the metabolic changes in U87 cells induced with chlorotoxin at different incubation time points by measuring the ratios of integrated areas corresponding to different chemical conformations of proteins, lipids, carbohydrates, and nucleic acids. Our results revealed spectral changes in U87 cells at different stages of incubation with chlorotoxin. The most notable change occurred after 30 minutes of incubation, where the band assigned to CH3 bending of lipid membranes shifted from 1456 cm-1 to 1450 cm-1. Consequently, a new shoulder appeared at wavenumber of 1466 cm-1 assigned to CH2 bending of lipid membranes for U87 cells treated with chlorotoxin. These changes indicate a direct interaction due to the presence of a molecular binding site between chlorotoxin and the lipids of the cell membrane. Another significant alternation occurred after 1 hour of incubation of U87 cells with chlorotoxin in the spectral region assigned for nucleic acids. This interaction included a large upshift of the band at 1036 cm-1 to 1051 cm-1 assigned to C-O stretching vibrations of ribose ring in RNA due to the hydrogen bonding with chlorotoxin. These findings suggest at least two different interactions of chlorotoxin with U87 human glioma cells that can be further explored for improving chlorotoxin containing drug delivery systems. We are currently using the ATR-FTIR method to conduct control experiments by measuring possible interactions of chlorotoxin with normal human astrocytes. Citation Format: Rana Falahat, Marzenna Wiranowska, Ryan Toomey, Norma Alcantar. Interactions of glioma cells with chlorotoxin: an ATR-FTIR spectroscopy study. [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 3909.

Dicationic Surfactants with Glycine Counter Ions for Oligonucleotide Transportation.

Gemini surfactants are good candidates to bind, protect, and deliver nucleic acids. Herein, the concept of amino acids (namely glycine) as counter ions of gemini surfactants for gene therapy application was explored. This study was conducted on DNA and RNA oligomers and two quaternary bis-imidazolium salts, having 2,5-dioxahexane and 2,8-dioxanonane spacer groups. The toxicity level of surfactants was assessed by an MTT assay, and their ability to bind nucleic acids was tested through electrophoresis. The nucleic acid conformation was established based on circular dichroism and infrared spectroscopic analyses. The structures of the formed complexes were characterized by small-angle scattering of synchrotron radiation. Both studied surfactants appear to be suitable for gene therapy; however, although they vary by only three methylene groups in the spacer, they differ in binding ability and toxicity. The tested oligonucleotides maintained their native conformations upon surfactant addition and the studied lipoplexes formed a variety of structures. In systems based on a 2,5-dioxahexane spacer, a hexagonal phase was observed for DNA-surfactant complexes and a micellar phase was dominant with RNA. For the surfactant with a 2,8-dioxanonane spacer group, the primitive cubic phase prevailed.

Molecular dynamics simulations reveal the balance of forces governing the formation of a guanine tetrad-a common structural unit of G-quadruplex DNA.

G-quadruplexes (G4) are nucleic acid conformations of guanine-rich sequences, in which guanines are arranged in the square-planar G-tetrads, stacked on one another. G4 motifs form in vivo and are implicated in regulation of such processes as gene expression and chromosome maintenance. The structure and stability of various G4 topologies were determined experimentally; however, the driving forces for their formation are not fully understood at the molecular level. Here, we used all-atom molecular dynamics to probe the microscopic origin of the G4 motif stability. By computing the free energy profiles governing the dissociation of the 3′-terminal G-tetrad in the telomeric parallel-stranded G4, we examined the thermodynamic and kinetic stability of a single G-tetrad, as a common structural unit of G4 DNA. Our results indicate that the energetics of guanine association alone does not explain the overall stability of the G-tetrad and that interactions involving sugar–phosphate backbone, in particular, the constrained minimization of the phosphate–phosphate repulsion energy, are crucial in providing the observed enthalpic stabilization. This enthalpic gain is largely compensated by the unfavorable entropy change due to guanine association and optimization of the backbone topology.

Structural Characterization of dsDNA Nanocomplexes based on Ammonium Gemini Surfactants

One of the most intensively studied groups of chemical compounds are gemini surfactants, because of their specific and attractive properties, especially the capability of stable binding of the nucleic acids, formation of spatial structures with high symmetry and relatively low cytotoxicity. They have found a broad range of applications in medicine, chemical technology, pharmaceutical industry [1]. These complexes can be applied as nonviral transfection delivery systems in gene therapy, a novel and most promising method for the treatment of a broad range of genetic diseases. Dicationic surfactants allow introduction of a transgene without inducing natural immunological response, and release it undamaged inside the cell. However, the successful completion of this process is difficult, because of the need for many studies in order to determine all necessary parameters [2-5].In our study we analysed nanocomplexes of cationic gemini surfactants (1,n-dodecanebis[(oxymethyl)alkyldimethylammonium]chlorides) with DNA oligomers using synchrotron radiation of small angle X-ray scattering, circular dichroism spectroscopy, agarose gel electrophoresis, polarization microscopy and transmission electron microscopy. We also analysed the cytotoxicity of our systems using MTT colorimetric assay on model cell cultures.We observed the formation of complexes, their morphology, spatial nanostructure, conformation of nucleic acid inside them and influence on the cells. The systems studied were found to be stable and the process of their formation was reproducible.This research project was financed by National Science Centre (Poland), decision no.DEC-2011/01/B/ST5/00846.[1] R. Zielinski, Surfactants - structure, properties, applications, WUP, 2009, Poznan.[2] P. C. Bell, et al. J. Am. Chem. Soc. 125 (2003) 1551-1558.[3] W.C. Brunner, M.F. Maestre Biolpolym.13 (1974) 345-357.[4] D. Simberg, D. et al., J. Biol. Chem. 276 (2001) 47453-47459.[5] S.D. Wetting, et al., J. Gene Med. 9 (2007) 649-658.

The structural stability and catalytic activity of DNA and RNA oligonucleotides in the presence of organic solvents.

Organic solvents and apolar media are used in the studies of nucleic acids to modify the conformation and function of nucleic acids, to improve solubility of hydrophobic ligands, to construct molecular scaffolds for organic synthesis, and to study molecular crowding effects. Understanding how organic solvents affect nucleic acid interactions and identifying the factors that dominate solvent effects are important for the creation of oligonucleotide-based technologies. This review describes the structural and catalytic properties of DNA and RNA oligonucleotides in organic solutions and in aqueous solutions with organic cosolvents. There are several possible mechanisms underlying the effects of organic solvents on nucleic acid interactions. The reported results emphasize the significance of the osmotic pressure effect and the dielectric constant effect in addition to specific interactions with nucleic acid strands. This review will serve as a guide for the selection of solvent systems based on the purpose of the nucleic acid-based experiments.

Spectrochemical analysis of sycamore (Acer pseudoplatanus) leaves for environmental health monitoring.

Terrestrial plants are ideal sentinels of environmental pollution, due to their sedentary nature, abundance and sensitivity to atmospheric changes. However, reliable and sensitive biomarkers of exposure have hitherto been difficult to characterise. Biospectroscopy offers a novel approach to the derivation of biomarkers in the form of discrete molecular alterations detectable within a biochemical fingerprint. We investigated the application of this approach for the identification of biomarkers for pollution exposure using the common sycamore (Acer pseudoplatanus) as a sentinel species. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy was used to interrogate leaf tissue collected from three sites exposed to different levels of vehicle exhaust emissions. Following multivariate analysis of acquired spectra, significant biochemical alterations were detected between comparable leaves from different sites that may constitute putative biomarkers for pollution-induced stress. These included differences in carbohydrate and nucleic acid conformations, which may be indicative of sub-lethal exposure effects. We also observed several corresponding spectral alterations in both the leaves of A. pseudoplatanus exposed to ozone pollution under controlled environmental conditions and in leaves infected with the fungal pathogen Rhytisma acerinum, indicating that some stress-induced changes are conserved between different stress signatures. These similarities may be indicative of stress-induced reactive oxygen species (ROS) generation, although further work is needed to verify the precise identity of infrared biomarkers and to identify those that are specific to pollution exposure. Taken together, our data clearly demonstrate that biospectroscopy presents an effective toolkit for the utilisation of higher plants, such as A. pseudoplatanus, as sentinels of environmental pollution.