Helical pyrene-array along the outside of duplex RNA

A helical pyrene-array was formed by incorporation of multiple pyrenes into the sugar residues of duplex RNA leading to strong excimer fluorescence.     

7-Deazapurine and 8-aza-7-deazapurine nucleoside and oligonucleotide pyrene "click" conjugates: synthesis, nucleobase controlled fluorescence quenching, and duplex stability

7-Deazapurine and 8-aza-7-deazapurine nucleosides related to dA and dG bearing 7-octadiynyl or 7-tripropargylamine side chains as well as corresponding oligonucleotides were synthesized. "Click" conjugation with 1-azidomethyl pyrene (10) resulted in fluorescent derivatives. Octadiynyl conjugates show only monomer fluorescence, while the proximal alignment of pyrene residues in the tripropargylamine derivatives causes excimer emission. 8-Aza-7-deazapurine pyrene "click" conjugates exhibit fluorescence emission much higher than that of 7-deazapurine derivatives. They are quenched by intramolecular charge transfer between the nucleobase and the dye. Oligonucleotide single strands decorated with two "double clicked" pyrenes show weak or no excimer fluorescence. However, when duplexes carry proximal pyrenes in complementary strands, strong excimer fluorescence is observed. A single replacement of a canonical nucleoside by a pyrene conjugate stabilizes the duplex substantially, most likely by stacking interactions: 6-12 °C for duplexes with a modified "adenine" base and 2-6 °C for a modified "guanine" base. The favorable photophysical properties of 8-aza-7-deazapurine pyrene conjugates improve the utility of pyrene fluorescence reporters in oligonucleotide sensing as these nucleoside conjugates are not affected by nucleobase induced quenching.     

Highly fluorescent conjugated pyrenes in nucleic acid probes: (phenylethynyl)pyrenecarbonyl-functionalized locked nucleic acids

In recent years, fluorescently labeled oligonucleotides have become a widely used tool in diagnostics, DNA sequencing, and nanotechnology. The recently developed (phenylethynyl)pyrenes are attractive dyes for nucleic acid labeling, with the advantages of long-wave emission relative to the parent pyrene, high fluorescence quantum yields, and the ability to form excimers. Herein, the synthesis of six (phenylethynyl)pyrene-functionalized locked nucleic acid (LNA) monomers M(1)-M(6) and their incorporation into DNA oligomers is described. Multilabeled duplexes display higher thermal stabilities than singly modified analogues. An increase in the number of phenylethynyl substituents attached to the pyrene results in decreased binding affinity towards complementary DNA and RNA and remarkable bathochromic shifts of absorption/emission maxima relative to the parent pyrene fluorochrome. This bathochromic shift leads to the bright fluorescence colors of the probes, which differ drastically from the blue emission of unsubstituted pyrene. The formation of intra- and interstrand excimers was observed for duplexes that have monomers M(1)-M(6) in both complementary strands and in numerous single-stranded probes. If more phenylethynyl groups are inserted, the detected excimer signals become more intense. In addition, (phenylethynyl)pyrenecarbonyl-LNA monomers M(4), M(5), and M(6) proved highly useful for the detection of single mismatches in DNA/RNA targets.     

Helically Assembled Pyrene Arrays on an RNA Duplex That Exhibit Circularly Polarized Luminescence with Excimer Formation

Circularly polarized luminescence (CPL) was observed in pyrene zipper arrays helically arranged on an RNA duplex. Hybridization of complementary RNA strands having multiple (two to five) 2′‐O‐pyrenylmethyl modified nucleosides affords an RNA duplex with normal thermal stability. The pyrene fluorophores are assembled like a zipper in a well‐defined helical manner along the axis of RNA duplex, which, upon 350 nm UV illumination, resulted in CPL emission with pyrene excimer formation. CPL (g_lum) levels observed for the pyrene arrays in dilute aqueous solution were +2×10^?2–+3.5×10^?2, which are comparable with |g_lum| for chiral organic molecules and related systems. The positive CPL signals are consistent with a right‐handed helical structure. Temperature dependence on CPL emission indicates that the stable rigid RNA structure is responsible for the strong CPL signals. The single pyrene‐modified RNA duplex did not show any CPL signal.     

Pyrene-stacked nanostructures constructed in the recombinant tobacco mosaic virus rod scaffold

The effect of pyrenes introduced into a tobacco mosaic virus (TMV) coat protein monomer on the formation and stability of the TMV assembly was investigated. The possible arrangement of the pyrenes in the inner cavity of the TMV rod was also estimated. The pyrene derivative was introduced to four specific amino acids in the cavity of the TMV rod structure. Rod-structure formation was examined by atomic force microscopy (AFM). Two pyrene-attached mutants (positions 99 and 100) assembled to increase the length of the rod structures by 2.5 microm at pH 5.5. The interaction of the pyrene moieties in the TMV cavity was investigated by steady-state and time-resolved spectroscopic analysis. Strong excimer emission with significantly short wavelength (465 nm) was observed from the two mutants mentioned above. Excitation and UV-visible spectra indicate that the pyrene moieties form pi-stacked structures in the TMV cavity. Details of the pyrene interaction were investigated by analyzing the fluorescence lifetime of the excimer. Results suggest that the pyrenes formed preassociated rigid structures with partially overlapped geometry in the restricted space of the TMV cavity. The pyrenes effectively stabilize the TMV rod through a pi-stacking interaction in a well-ordered way, and the single pyrene moiety introduced into the monomer affects the overall formation of the TMV rod structure.     

Color-tunable fluorescent organogels: columnar self-assembly of pyrene-containing oligo(glutamic acid)s

New fluorescent gelators containing pyrene moieties and dendritic oligopeptides have been developed. These molecules self-assemble into 1D helical columnar structures that lead to the formation of 3D fibrous random networks. The resulting gel materials show monomer emission of pyrenes because the hydrogen-bonded array of the oligopeptide moieties greatly suppresses the formation of pyrene excimers. In contrast, in the sol states green excimer emission of the pyrenes is observed because of the dissociation of intermolecular hydrogen bonds. This is the first example of the reverse-mode color switching of fluorescence for supramolecular pyrene assemblies.     

Syntheses and fluorescence of RNA conjugates having pyrene-modified adenosine and nitrobenzene-modified uridine base pairs

We prepared RNA duplexes possessing 2′-O-(1-pyrenylmethyl)adenosine and 5-(4-nitrophenyl)uridine base pairs. In the duplexes, pyrene serves as a photo-excitable electron donor and 5-(4-nitrophenyl)uridine acts as an electron acceptor. The donor-acceptor-modified RNA duplexes showed very weak fluorescence originating from the pyrene monomer and excimer emissions, which occur due to electron transfer from the excited pyrene to the nitrobenzene acceptor.     

Chirality of Anisotropic Metal Nanowires with a Distinct Multihelix

Two‐dimensional (2D) anisotropic silver nanowire (AgNW) arrays, fabricated inside chiral mesoporous silica (CMS), exhibited strong and tunable plasmon circular dichroism (CD) signals in the visible and near‐IR regions due to collective dipole coupling between the anisotropic AgNWs. The multihelix with a helical channel orientation and helical arrays of opposite handedness in CMS played a predominant effects on the transversal and longitudinal chirality of the AgNWs, respectively.This behavior differs from both isotropic‐nanoparticle and single‐helix‐induced CD responses. This system will provide new insight into the optical activity of metal inorganic nanoparticles capped with chiral organic molecules and assembled in chiral environments.     

2-Aminopurine: a probe of structural dynamics and charge transfer in DNA and DNA:RNA hybrids

Spectroscopic techniques are employed to probe relationships between structural dynamics and charge transfer (CT) efficiency in DNA duplexes and DNA:RNA hybrids containing photoexcited 2-aminopurine (Ap). To better understand the variety of interactions and reactions, including CT, between Ap and DNA, the fluorescence behavior of Ap is investigated in a full series of redox-inactive as well as redox-active assemblies. Thus, Ap is developed as a dual reporter of structural dynamics and base-base CT reactions in nucleic acid duplexes. CD, NMR, and thermal denaturation profiles are consistent with the family of DNA duplexes adopting a distinct conformation versus the DNA:RNA hybrids. Fluorescence measurements establish that the d(A)-r(U) tract of the DNA:RNA hybrid exhibits enhanced structural flexibility relative to that of the d(A)-d(T) tract of the DNA duplexes. The yield of CT from either G or 7-deazaguanine (Z) to Ap in the assemblies was determined by comparing Ap emission in redox-active G- or Z-containing duplexes to otherwise identical duplexes in which the G or Z is replaced by inosine (I), the redox-inactive nucleoside analogue. Investigations of CT not only demonstrate efficient intrastrand base-base CT in the DNA:RNA hybrids but also reveal a distance dependence of CT yield that is more shallow through the d(A)-r(U) bridge of the A-form DNA:RNA hybrids than through the d(A)-d(T) bridge of the B-form DNA duplexes. The shallow distance dependence of intrastrand CT in DNA:RNA hybrids correlates with the increased conformational flexibility of bases within the hybrid duplexes. Measurements of interstrand base-base CT provide another means to distinguish between the A- and B-form helices. Significantly, in the A-form DNA:RNA hybrids, a similar distance dependence is obtained for inter- and intrastrand reactions, while, in B-DNA, a more shallow distance dependence is evident with interstrand CT reactions. These observations are consistent with evaluations of intra- and interstrand base overlap in A- versus B-form duplexes. Overall, these data underscore the sensitivity of CT chemistry to nucleic acid structure and structural dynamics.     

Structure of the alpha-homo-DNA:RNA duplex and the function of twist and slide to catalogue nucleic acid duplexes

High-resolution NMR studies of an alpha-homo-DNA:RNA duplex reveal the formation of a right-handed parallel-oriented helix. It differs significantly from a standard A- or B-type helix by a small twist value (26.2 degrees ), which leads to a helical pitch of 13.7 base pairs per helical turn, a negative inclination (-1.78 Angstrom) and a large x displacement (5.90 Angstrom). The rise (3.4 Angstrom) is similar to that found in B-DNA. The solution of this new helix structure has stimulated us to develop a mathematical and geometrical model based on slide and twist parameters to describe nucleic acid duplexes. All existing duplexes can be positioned within this landscape, which can be used to understand the helicalization process.     

Supramolecular helical porphyrin arrays using DNA as a scaffold

A diphenyl porphyrin substituted nucleotide was incorporated site specifically into DNA, leading to helical stacked porphyrin arrays in the major groove of the duplexes. The porphyrins show an electronic interaction which is significantly enhanced compared to the analogous tetraphenyl porphyrin (TPP) as shown in the large exciton coupling of the porphyrin B-band absorbance. Analogous to the TPP-DNA, an induced helical secondary structure is observed in the single strand porphyrin-DNA. The modified DNA can be hybridised to an immobilised complementary strand leading to fluorescent beads.     

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.     

Atomic detail investigation of the structure and dynamics of DNA.RNA hybrids: a molecular dynamics study

DNA.RNA hybrid duplexes are biologically important molecules and are shown to have potential therapeutic properties. To investigate the relationship between structures, energetics, solvation and RNase H activity of hybrid duplexes in comparison with pure DNA and RNA duplexes, a molecular dynamics study using the CHARMM27 force field was undertaken. The structural properties of all four nucleic acids considered are in very good agreement with the experimental data. The backbone dihedral angles and the puckering of the (deoxy)ribose indicate that the purine rich strands retain their A-/B-like properties but the pyrimidine rich DNA strand undergoes A-B conformational transitions. The minor groove widths of the hybrid structures are narrower than those in the RNA duplex, a requirement for RNase H binding. In addition, sampling of noncanonical phosphodiester backbone dihedrals by the DNA strands, differential solvation properties and helical properties, most notably rise, are suggested to contribute to hybrids being RNase H substrates. Differential RNase H activity toward hybrids containing purine versus pyrimidine rich RNA strands is suggested to be due to sampling of values of the phosphodiester backbone dihedrals in the DNA strands. Notably, the present results indicate that hybrids have decreased flexibility as compared to RNA, in contrast to previous reports.     

Theoretical analysis of antisense duplexes: determinants of the RNase H susceptibility

The structure and dynamic properties of different antisense related duplexes (DNA x RNA, 2'O-Me-DNA x RNA, 2'F-ANA x RNA, C5(Y)-propynyl-DNA x RNA, ANA x RNA, and control duplexes DNA x DNA and RNA x RNA) have been determined by means of long molecular dynamics simulations (covering more than 0.5 micros of fully solvated unrestrained MD simulation). The massive analysis presented here allows us to determine the subtle differences between the different duplexes, which in all cases pertain to the same structural family. This analysis provides information on the molecular determinants that allow RNase H to recognize and degrade some of these duplexes, whereas others with apparently similar conformations are not affected. Subtle structural and deformability features define the key properties used by RNase H to discriminate between duplexes.     

(SR/RS)-cyclohexanyl PNAs: conformationally preorganized PNA analogues with unprecedented preference for duplex formation with RNA

PNA oligomers H-GTAGATCAT-lys-NH2 with cis-(1S,2R/1R,2S)-cyclohexyl-T (III) in the backbone form PNA:RNA duplexes with Tm approximately 30-50 degrees C higher than that of PNA:DNA duplexes. In comparison, cis-(1S,2R/1R,2S)-cyclopentyl PNA-T (IV) form highly stable duplexes with both RNA and DNA without discrimination.     

Multilabeled pyrene-functionalized 2'-amino-LNA probes for nucleic acid detection in homogeneous fluorescence assays

Homogeneous fluorescence assays for detection of nucleic acids are widely used in biological sciences. Typically, probes such as molecular beacons that rely on distance-dependent fluorescence quenching are used for such assays. Less attention has been devoted to tethering a single kind of fluorophores to oligonucleotides and exploiting hybridization-induced modulation of fluorescence intensity for nucleic acid detection. Herein, thermal denaturation experiments and fluorescence properties of oligodeoxyribonucleotides containing one or more 2'-N-(pyren-1-yl)carbonyl-2'-amino-LNA monomer(s) X are described. These pyrene-functionalized 2'-amino-LNAs display large increases in thermal stability against DNA/RNA complements with excellent Watson-Crick mismatch discrimination. Upon duplex formation of appropriately designed 2'-N-(pyren-1-yl)carbonyl-2'-amino-LNA probes and complementary DNA/RNA, intensive fluorescence emission with quantum yields between 0.28 and 0.99 are observed. Quantum yields of such magnitudes are unprecedented among pyrene-labeled oligonucleotides. Molecular modeling studies suggest that the dioxabicyclo[2.2.1]heptane skeleton and amide linkage of monomer X fix the orientation of the pyrene moiety in the minor groove of a nucleic acid duplex. Interactions between pyrene and nucleobases, which typically lead to quenching of fluorescence, are thereby reduced. Duplexes between multiple modified probes and DNA/RNA complements exhibit additive increases in fluorescence intensity, while the fluorescence of single stranded probes becomes increasingly quenched. Up to 69-fold increase in fluorescence intensity (measured at lambda(em) = 383 nm) is observed upon hybridization to DNA/RNA. The emission from duplexes of multiple modified probes and DNA/RNA at concentrations down to less than 500 nM can easily be seen by the naked eye using standard illumination intensities.     

Duplex formation and the onset of helicity in poly d(CG)n oligonucleotides in a solvent-free environment

The gas-phase conformations of a series of cytosine/guanine DNA duplexes were examined by ion mobility and molecular dynamics methods. Deprotonated duplex ions were formed by electrospray ionization, and their collision cross sections measured in helium were compared to calculated cross sections of theoretical models generated by molecular dynamics. The 4-mer (dCGCG) and 6-mer (dCGCGCG) duplexes were found to have globular conformations. Globular and helical structures were observed for the 8-mer (dCGCGCGCG) duplex, with the globular form being the more favored conformer. For the 10-mer (dCGCGCGCGCG), 14-mer (dCGCGCGCGCGCGCG), and 18-mer (dCGCGCGCGCGCGCGCGCG) duplexes, only helical structures were observed in the ion mobility measurements. Theory predicts that the helical structures are less stable than the globular forms in the gas phase and should collapse into the globular form given enough time. However, molecular dynamics simulations at 300 K indicate the helical structures are stable in aqueous solution and will retain their conformations for a limited time in the gas phase. The presence of helical structures in the ion mobility experiments indicates that the duplexes retain "solution structures" in the gas phase on the millisecond time scale.     

Supramolecular p-n-heterojunctions by co-self-organization of oligo(p-phenylene vinylene) and perylene bisimide dyes

Comparative studies on hydrogen-bonded versus covalently linked donor-acceptor-donor dye arrays obtained from oligo(p-phenylene vinylene)s (OPVs) as donor and bay-substituted perylene bisimides (PERYs) as acceptor dyes are presented. Both systems form well-ordered J-type aggregates in methylcyclohexane, but only hydrogen-bonded arrays afford hierarchically assembled chiral OPV-PERY dye superstructures consisting of left-handed helical pi-pi co-aggregates (CD spectroscopy) of the two dyes that further assemble into right-handed nanometer-scale supercoils in the solid state (AFM study). In the case of hydrogen-bonded arrays, the stability of the aggregates in solution increases with increasing conjugation length of the OPV unit. The well-defined co-aggregated dyes presented here exhibit photoinduced electron transfer on subpicosecond time scale, and thus, these supramolecular entities might serve as valuable nanoscopic functional units.     

Studies on the Conformation of Poly(1-naphthol) Assembled by Horseradish Peroxidase in Aqueous Micelle

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A label-free fluorescence sensor for probing the interaction of oligonucleotides with target molecules

This paper describes a universal, label-free fluorescence sensor that gauges the interaction of oligonucleotides with their targets. The sensor is based on supramolecular assemblies formed by oligonucleotides (polyanions) and small molecule cation surfactant in aqueous solution. The environmentally dependent dye pyrene, encapsulated in the apolar interiors of the assemblies via hydrophobic interactions works as the fluorescence probe. Target binding causes the conformational change of the oligonucleotides, which results in disorganization of supramolecular assemblies, release of pyrene into the aqueous solution and subsequent quenching of its fluorescence. The kinetic processes (including the formation of supramolecular assemblies and the release of pyrenes after adding the targets) were investigated. The fluorescence decreases of pyrenes are proportional to the concentrations of targets within the linear ranges. This label-free fluorescence system is simple, convenient, low cost, and can serve as an alternative tool for interaction studies of oligonucleotides with their targets, especially with small molecular targets.