Modulation of pyrene fluorescence in DNA probes depends upon the nature of the conformationally restricted nucleotide

The DNA probes (ODNs) containing a 2'-N-(pyren-1-yl)-group on the conformationally locked nucleosides [2'-N-(pyren-1-yl)carbonyl-azetidine thymidine, Aze-pyr (X), and 2'-N-(pyren-1-yl)carbonyl-aza-ENA thymidine, Aza-ENA-pyr (Y)], show that they can bind to complementary RNA more strongly than to the DNA. The Aze-pyr (X) containing ODNs with the complementary DNA and RNA duplexes showed an increase in the fluorescence intensity (measured at lambda em approximately 376 nm) depending upon the nearest neighbor at the 3'-end to X [dA ( approximately 12-20-fold) > dG ( approximately 9-20-fold) > dT ( approximately 2.5-20-fold) > dC ( approximately 6-13-fold)]. They give high fluorescence quantum yields (Phi F = 0.13-0.89) as compared to those of the single-stranded ODNs. The Aza-ENA-pyr (Y)-modified ODNs, on the other hand, showed an enhancement of the fluorescence intensity only with the complementary DNA (1.4-3.9-fold, Phi F = 0.16-0.47); a very small increase in fluorescence is also observed with the complementary RNA (1.1-1.7-fold, Phi F = 0.17-0.22), depending both upon the site of the Y modification introduced as well as on the chemical nature of the nucleobase adjacent to the modification site into the ODN. The fluorescence properties, thermal denaturation experiments, absorption, and circular dichroism (CD) studies with the X- and Y-modified ODNs in the form of matched homo- and heteroduplexes consistently suggested (i) that the orientation of the pyrene moiety is outside the helix of the nucleic acid duplexes containing a dT-d/rA base pair at the 3'-end of the modification site for both X and Y types of modifications, and (ii) that the microenvironment around the pyrene moiety in the ODN/DNA and ODN/RNA duplexes is dictated by the chemical nature of the conformational constraint in the sugar moiety, as well as by the nature of neighboring nucleobases. The pyrene fluorescence emission in both X and Y types of the conformationally restricted nucleotides is found to be sensitive to a mismatched base present in the target RNA: (i) The X-modified ODN showed a decrease ( approximately 37-fold) in the fluorescence intensity (measured at lambda em approximately 376 nm) upon duplex formation with RNA containing a G nucleobase mismatch (dT-rG pair instead of dT-rA) opposite to the modification site. (ii) In contrast, the Y-modified ODN in the heteroduplex resulted in a approximately 3-fold increase in the fluorescence intensity upon dT-rG mismatch, instead of matched dT-rA pair, in the RNA strand. Our data corroborate that the pyrene moiety is intercalated in the X-modified mismatched ODN/RNA (G mismatch) heteroduplex as compared to that of the Y-modified ODN/RNA (G mismatch) heteroduplex, in which it is located outside the helix.     

Characterization of G‐Quadruplex/Hairpin Transitions of RNAs by 19F NMR Spectroscopy

2′‐O‐[(4‐Trifluoromethyl‐triazol‐1‐yl)methyl] reporter groups have been incorporated into guanosine‐rich RNA models (including a known bistable Qd/Hp RNA and two G‐rich regions of mRNA of human prion protein, PrP) and applied for the ¹⁹F NMR spectroscopic characterization of plausible G‐quadruplex/hairpin (Qd/Hp) transitions in these RNA structures. For the synthesis of the CF₃‐labeled RNAs, phosphoramidite building blocks of 2′‐O‐[(4‐CF₃‐triazol‐1‐yl)methyl] nucleosides (cytidine, adenosine, and guanosine) were prepared and used as an integral part of the standard solid‐phase RNA synthesis. The obtained ¹⁹F NMR spectra supported the usual characterization data (obtained by UV‐ and CD‐melting profiles and by ¹H NMR spectra of the imino regions) and additionally gave more detailed information on the Qd/Hp transitions. The molar fractions of the secondary structural species (Qd, Hp) upon thermal denaturation and under varying ionic conditions could be determined from the intensities and shifts of the ¹⁹F NMR signals. For a well‐behaved Qd/Hp transition, thermodynamic parameters could be extracted.     

Aromatic 19F–13C TROSY—[19F, 13C]‐Pyrimidine Labeling for NMR Spectroscopy of RNA

We present the access to [5‐¹⁹F, 5‐¹³C]‐uridine and ‐cytidine phosphoramidites for the production of site‐specifically modified RNAs up to 65 nucleotides (nts). The amidites were used to introduce [5‐¹⁹F, 5‐¹³C]‐pyrimidine labels into five RNAs—the 30 nt human immunodeficiency virus trans activation response (HIV TAR) 2 RNA, the 61 nt human hepatitis B virus ? (hHBV ?) RNA, the 49 nt SAM VI riboswitch aptamer domain from B. angulatum, the 29 nt apical stem loop of the pre‐microRNA (miRNA) 21 and the 59 nt full length pre‐miRNA 21. The main stimulus to introduce the aromatic ¹⁹F–¹³C‐spin topology into RNA comes from a work of Boeszoermenyi et al., in which the dipole‐dipole interaction and the chemical shift anisotropy relaxation mechanisms cancel each other leading to advantageous TROSY properties shown for aromatic protein sidechains. This aromatic ¹³C–¹⁹F labeling scheme is now transferred to RNA. We provide a protocol for the resonance assignment by solid phase synthesis based on diluted [5‐¹⁹F, 5‐¹³C]/[5‐¹⁹F] pyrimidine labeling. For the 61 nt hHBV ? we find a beneficial ¹⁹F–¹³C TROSY enhancement, which should be even more pronounced in larger RNAs and will facilitate the NMR studies of larger RNAs. The [¹⁹F, ¹³C]‐labeling of the SAM VI aptamer domain and the pre‐miRNA 21 further opens the possibility to use the biorthogonal stable isotope reporter nuclei in in vivo NMR to observe ligand binding and microRNA processing in a biological relevant setting.     

Specific Detection and Imaging of Enzyme Activity by Signal‐Amplifiable Self‐Assembling 19F MRI Probes

Specific turn‐on detection of enzyme activities is of fundamental importance in drug discovery research, as well as medical diagnostics. Although magnetic resonance imaging (MRI) is one of the most powerful techniques for noninvasive visualization of enzyme activity, both in vivo and ex vivo, promising strategies for imaging specific enzymes with high contrast have been very limited to date. We report herein a novel signal‐amplifiable self‐assembling ¹⁹F NMR/MRI probe for turn‐on detection and imaging of specific enzymatic activity. In NMR spectroscopy, these designed probes are “silent” when aggregated, but exhibit a disassembly driven turn‐on signal change upon cleavage of the substrate part by the catalytic enzyme. Using these ¹⁹F probes, nanomolar levels of two different target enzymes, nitroreductase (NTR) and matrix metalloproteinase (MMP), could be detected and visualized by ¹⁹F NMR spectroscopy and MRI. Furthermore, we have succeeded in imaging the activity of endogenously secreted MMP in cultured media of tumor cells by ¹⁹F MRI, depending on the cell lines and the cellular conditions. These results clearly demonstrate that our turn‐on ¹⁹F probes may serve as a screening platform for the activity of MMPs.     

Parahydrogen‐Induced Polarization Transfer to 19F in Perfluorocarbons for 19F NMR Spectroscopy and MRI

Fluorinated substances are important in chemistry, industry, and the life sciences. In a new approach, parahydrogen‐induced polarization (PHIP) is applied to enhance ¹⁹F MR signals of (perfluoro‐n‐hexyl)ethene and (perfluoro‐n‐hexyl)ethane. Unexpectedly, the end‐standing CF₃ group exhibits the highest amount of polarization despite the negligible coupling to the added protons. To clarify this non‐intuitive distribution of polarization, signal enhancements in deuterated chloroform and acetone were compared and ¹⁹F–¹⁹F NOESY spectra, as well as ¹⁹F T₁ values were measured by NMR spectroscopy. By using the well separated and enhanced signal of the CF₃ group, first ¹⁹F MR images of hyperpolarized linear semifluorinated alkenes were recorded.     

Comparative Electrochemical and Spectroscopic Studies of I‐Motif‐forming DNA Nonamers

The paper shows the structural diversity of cytosine (C)‐rich oligodeoxynucleotides (ODNs) arising from their detail nucleotide sequence and experimental conditions. In slightly acidic solutions, the ODN nonamers with different adenine (A) and cytosine (C) sequences can adopt non‐canonical structures involving protonated bases. A distinct secondary structure formed in (C)‐rich sequences, called i‐motif (iM), consists of hemiprotonated and intercalated cytosine base pairs (C.C⁺). Folding and unfolding of particular structures in solutions were monitored by ¹H NMR and CD spectroscopies and native polyacrylamide gel electrophoresis (PAGE), which are capable to determine their structural characteristics. Effects of sequences and their proclivity to formation of the iM on electrochemical behaviour of the ODN nonamers were studied by electrochemical methods. The LSV signals of A and C obtained from the reductive dissolution of ODN adsorption layers on a hanging mercury drop electrode were processed by elimination voltammetry with linear scan (EVLS), which revealed complex effects of the nonamer properties (namely their primary and secondary structure confirmed in solution) on their adsorption and reduction activity.     

19F‐MAS NMR on Proteorhodopsin: Enhanced Protocol for Site‐Specific Labeling for General Application to Membrane Proteins†

Proteorhodopsin (PR) is a light‐driven proton pump found in near‐surface marine γ‐proteobacteria. The green absorbing variant has three cysteines at positions 107, 156 and 175. We probed the accessibility of these residues by ¹⁹F‐MAS NMR. For this purpose, an efficient but simple protocol for chemical fluorine labeling of accessible cysteines in membrane proteins was established. This one‐step reaction was applied to detergent‐solubilized PR before reconstitution into phospholipids. All three cysteines could be labeled and showed distinct ¹⁹F chemical shifts with different integral intensities. The accessibility of these cysteines is discussed in the context of a homology model. With the chemical cysteine labeling procedure shown here, an attractive option for site‐directed solid‐state NMR studies on other membrane proteins is offered due to the high intrinsic sensitivity of ¹⁹F‐MAS NMR.     

C4’-Fluorinated Oligodeoxynucleotides: Synthesis,Stability, Structural Studies

Fluoro-substitution on the ribose moiety (e. g., 2’-F-deoxyribonucleotide) represents a popular way to modulate the ribose conformation and, hence, the structure and function of nucleic acids. In the present study, we synthesized 4’-F-deoxythymidine (^4’-FT) and introduced it to oligodeoxyribonucleotides (ODNs). Though scission of the glycosylic bond of ^4’-FT followed by strand cleavage occurred to some extent under alkaline conditions, the ^4’-FT-modified ODNs were rather stable in neutral buffers. NMR studies showed that like 2’-F-deoxyribonucleoside, ^4’-FT exists predominantly in the North conformation not only in the nucleoside form but also in the context of ODN strands. Circular dichroism spectroscopy, thermal denaturing and RNase H1 footprinting studies of ^4’-FT-modified ODN/cDNA and ODN/cRNA duplexes indicated that the North conformation tendency of ^4’-FT is maintained in the duplexes, leading to a local structural perturbation. Collectively, 4’-F-deoxyribonucleotide structurally resembles the 2’-F-deoxyribonucleotide but imparts less structural perturbation to the duplex than the latter.     

Fluorinated Carbohydrates as Lectin Ligands: Dissecting Glycan–Cyanovirin Interactions by Using 19F NMR Spectroscopy

NMR spectroscopy and isothermal titration calorimetry (ITC) are powerful methods to investigate ligand–protein interactions. Here, we present a versatile and sensitive fluorine NMR spectroscopic approach that exploits the ¹⁹F nucleus of ¹⁹F‐labeled carbohydrates as a sensor to study glycan binding to lectins. Our approach is illustrated with the 11 kDa Cyanovirin‐N, a mannose binding anti‐HIV lectin. Two fluoro‐deoxy sugar derivatives, methyl 2‐deoxy‐2‐fluoro‐α‐D ‐mannopyranosyl‐(1→2)‐α‐D ‐mannopyranoside and methyl 2‐deoxy‐2‐fluoro‐α‐D ‐mannopyranosyl‐(1→2)‐α‐D ‐mannopyranosyl‐(1→2)‐α‐D ‐mannopyranoside were utilized. Binding was studied by ¹⁹F NMR spectroscopy of the ligand and ¹H–¹⁵N HSQC NMR spectroscopy of the protein. The NMR data agree well with those obtained from the equivalent reciprocal and direct ITC titrations. Our study shows that the strategic design of fluorinated ligands and fluorine NMR spectroscopy for ligand screening holds great promise for easy and fast identification of glycan binding, as well as for their use in reporting structural and/or electronic perturbations that ensue upon interaction with a cognate lectin.     

Through‐space 19F–15N couplings for the assignment of stereochemistry in flubenzimine

Through‐space ¹⁹F–¹⁵N couplings revealed the configuration of flubenzimine, with the CF₃ group on N4 pointing towards the lone pair of N5. The ¹⁹F–¹⁵N coupling constants were measured at natural abundance using a spin‐state selective indirect‐detection pulse sequence. As ¹⁵N‐labelled proteins are routinely synthesized for NMR studies, through‐space ¹⁹F–¹⁵N couplings have the potential to probe the stereochemistry of these proteins by ¹⁹F labelling of some amino acids or can reveal the site of docking of fluorine‐containing drugs. Copyright © 2016 John Wiley & Sons, Ltd.     

Design,Synthesis, and Application of an Optimized Monofluorinated Aliphatic Label for Peptide Studies by Solid‐State 19F NMR Spectroscopy

A conformationally restricted monofluorinated α‐amino acid, (3‐fluorobicyclo[1.1.1]pentyl)glycine (F‐Bpg), was designed as a label for the structural analysis of membrane‐bound peptides by solid‐state ¹⁹F NMR spectroscopy. The compound was synthesized and validated as a ¹⁹F label for replacing natural aliphatic α‐amino acids. Calculations suggested that F‐Bpg is similar to Leu/Ile in terms of size and lipophilicity. The ¹⁹F NMR label was incorporated into the membrane‐active antimicrobial peptide PGLa and provided information on the structure of the peptide in a lipid bilayer.     

Design,Synthesis, and Application of an Optimized Monofluorinated Aliphatic Label for Peptide Studies by Solid‐State 19F NMR Spectroscopy

A conformationally restricted monofluorinated α‐amino acid, (3‐fluorobicyclo[1.1.1]pentyl)glycine (F‐Bpg), was designed as a label for the structural analysis of membrane‐bound peptides by solid‐state ¹⁹F NMR spectroscopy. The compound was synthesized and validated as a ¹⁹F label for replacing natural aliphatic α‐amino acids. Calculations suggested that F‐Bpg is similar to Leu/Ile in terms of size and lipophilicity. The ¹⁹F NMR label was incorporated into the membrane‐active antimicrobial peptide PGLa and provided information on the structure of the peptide in a lipid bilayer.     

The 3F Library: Fluorinated Fsp3‐Rich Fragments for Expeditious 19F NMR Based Screening

Fragment‐based drug discovery (FBDD) is a popular method in academia and the pharmaceutical industry for the discovery of early lead candidates. Despite its wide‐spread use, the approach still suffers from laborious screening workflows and a limited diversity in the fragments applied. Presented here is the design, synthesis, and biological evaluation of the first fragment library specifically tailored to tackle both these challenges. The 3F library of 115 fluorinated, Fsp³‐rich fragments is shape diverse and natural‐product‐like with desirable physicochemical properties. The library is perfectly suited for rapid and efficient screening by NMR spectroscopy in a two‐stage workflow of ¹⁹F NMR and subsequent ¹H NMR methods. Hits against four diverse protein targets are widely distributed among the fragment scaffolds in the 3F library and a 67 % validation rate was achieved using secondary assays. This collection is the first synthetic fragment library tailor‐made for ¹⁹F NMR screening and the results demonstrate that the approach should find broad application in the FBDD community.     

Specific Induced Circular Dichroism and Enhanced B to Z Transitions of Duplexes Stabilized by Chromophore‐Linked Alkynylnucleoside Residues

We have developed an induced circular dichroism (ICD) probe with a chromophore‐linked alkynyldeoxyribose skeleton for analyzing higher‐order structures of DNA duplexes in the visible‐light region. When CG‐repeated oligonucleotides (ODNs) with the probe at their 5′ ends adopted Z‐form duplexes at a high NaCl concentration, strong ICD signals were observed at the absorptive region of the chromophore. On the other hand, their B‐form duplexes, formed at a low NaCl concentration, produced a faint ICD signal. The specific ICD for the Z‐form duplexes was found to appear only when the chromophores were attached at the 5′ ends of each of the ODNs. Furthermore, the chromophoric alkynylnucleoside residues effectively promoted the B to Z transition of the ODN.     

Solution structure of a DNA duplex containing a guanine-difluorotoluene pair: a wobble pair without hydrogen bonding?

The incorporation of synthetic nucleoside analogues into DNA duplexes provides a unique opportunity to probe both structure and function of nucleic acids. We used 1H and 19F NMR and molecular dynamics calculations to determine the solution structures of two similar DNA decamer duplexes, one containing a central G-T mismatched or "wobble" base pair, and one in which the thymine in this base pair is replaced by difluorotoluene (a thymine isostere) creating a G-F pair. Here, we show that the non-hydrogen-bonding G-F pair stacks relatively well into the helix and that the distortions caused by each non-Watson-Crick G-T or G-F base pair are quite localized to a three base pair site around the mismatch. A detailed structural analysis reveals that the absence of hydrogen bonding introduces more dynamic motion into the G-F pair relative to G-T and permits the G-F pair to exhibit stacking and conformational features characteristic of both a Watson-Crick base pair (on the guanine containing strand) and a wobble base pair (on the strand containing the difluorotoluene). We used these results to posit a rationale for recognition and repair of mismatch sites in DNA.     

Chemical‐Shift‐Resolved 19F NMR Spectroscopy between 13.5 and 135 MHz: Overhauser–DNP‐Enhanced Diagonal Suppressed Correlation Spectroscopy

Overhauser–DNP‐enhanced homonuclear 2D ¹⁹F correlation spectroscopy with diagonal suppression is presented for small molecules in the solution state at moderate fields. Multi‐frequency, multi‐radical studies demonstrate that these relatively low‐field experiments may be operated with sensitivity rivalling that of standard 200–1000 MHz NMR spectroscopy. Structural information is accessible without a sensitivity penalty, and diagonal suppressed 2D NMR correlations emerge despite the general lack of multiplet resolution in the 1D ODNP spectra. This powerful general approach avoids the rather stiff excitation, detection, and other special requirements of high‐field ¹⁹F NMR spectroscopy.     

Assessing the Influence of Mutation on GTPase Transition States by Using X‐ray Crystallography, 19F NMR,and DFT Approaches

We report X‐ray crystallographic and ¹⁹F NMR studies of the G‐protein RhoA complexed with MgF₃⁻, GDP, and RhoGAP, which has the mutation Arg85′Ala. When combined with DFT calculations, these data permit the identification of changes in transition state (TS) properties. The X‐ray data show how Tyr34 maintains solvent exclusion and the core H‐bond network in the active site by relocating to replace the missing Arg85′ sidechain. The ¹⁹F NMR data show deshielding effects that indicate the main function of Arg85′ is electronic polarization of the transferring phosphoryl group, primarily mediated by H‐bonding to O^3G and thence to P^G. DFT calculations identify electron‐density redistribution and pinpoint why the TS for guanosine 5′‐triphosphate (GTP) hydrolysis is higher in energy when RhoA is complexed with RhoGAP_Arg85′Ala relative to wild‐type (WT) RhoGAP. This study demonstrates that ¹⁹F NMR measurements, in combination with X‐ray crystallography and DFT calculations, can reliably dissect the response of small GTPases to site‐specific modifications.     

Assessing the Influence of Mutation on GTPase Transition States by Using X‐ray Crystallography, 19F NMR,and DFT Approaches

We report X‐ray crystallographic and ¹⁹F NMR studies of the G‐protein RhoA complexed with MgF₃⁻, GDP, and RhoGAP, which has the mutation Arg85′Ala. When combined with DFT calculations, these data permit the identification of changes in transition state (TS) properties. The X‐ray data show how Tyr34 maintains solvent exclusion and the core H‐bond network in the active site by relocating to replace the missing Arg85′ sidechain. The ¹⁹F NMR data show deshielding effects that indicate the main function of Arg85′ is electronic polarization of the transferring phosphoryl group, primarily mediated by H‐bonding to O^3G and thence to P^G. DFT calculations identify electron‐density redistribution and pinpoint why the TS for guanosine 5′‐triphosphate (GTP) hydrolysis is higher in energy when RhoA is complexed with RhoGAP_Arg85′Ala relative to wild‐type (WT) RhoGAP. This study demonstrates that ¹⁹F NMR measurements, in combination with X‐ray crystallography and DFT calculations, can reliably dissect the response of small GTPases to site‐specific modifications.     

Delivering Structural Information on the Polar Face of Membrane‐Active Peptides: 19F‐NMR Labels with a Cationic Side Chain

Conformationally constrained non‐racemizing trifluoromethyl‐substituted lysine isosteres [(E)‐ and (Z)‐TCBLys] with charged side chains are presented as a new type of ¹⁹F‐NMR labels for peptide studies. Design of the labels, their synthesis, incorporation into peptides and experimental demonstration of their application for solid state NMR studies of membrane‐active peptides are described. A series of fluorine‐labeled analogues of the helical amphipathic antimicrobial peptide PGLa(Nle) was obtained, in which different lysine residues in the original peptide sequence were replaced, one at a time, by either (E)‐ or (Z)‐TCBLys. Antimicrobial activities of the synthesized analogues were practically the same as those of the parent peptide. The structural and orientational parameters of the helical PGLa(Nle) peptide in model bilayers, as determined using the novel labels confirmed and refined the previously known structure. (E)‐ and (Z)‐TCBLys, as a set of cationic ¹⁹F‐NMR labels, were shown to deliver structural information about the charged face of amphipathic peptides by solid state ¹⁹F‐NMR, previously inaccessible by this method.