Oligodeoxynucleotides containing palindrome sequences with internal 5'-CpG-3' act directly on human NK and activated T cells to induce IFN-gamma production in vitro.

Previous studies have shown that the action of bacterial or synthetic oligodeoxynucleotide (oligo-DNA) on mouse NK cells to produce IFN-gamma is mediated mostly by monocytes/macrophages activated by olig-DNA. However, its action on human IFN-gamma-producing cells has not been well investigated. In the present study, we examined the effect of oligo-DNAs on highly purified human NK and T cells. Bacillus Calmette-Guérin-derived or synthetic oligo-DNAs induced NK cells to produce IFN-gamma with an increased CD69 expression, and the autocrine IFN-gamma enhanced their cytotoxicity. The response of NK cells to oligo-DNAs was enhanced when the cells were activated with IL-2, IL-12, or anti-CD16 Ab. T cells did not produce IFN-gamma in response to oligo-DNAs but did respond independently of IL-2 when they were stimulated with anti-CD3 Ab. In the action of oligo-DNAs, the palindrome sequence containing unmethylated 5'-CpG-3' motif(s) appeared to play an important role in the IFN-gamma-producing ability of NK cells. The changes of base composition inside or outside the palindrome sequence altered its activity: The homooligo-G-flanked GACGATCGTC was the most potent IFN-gamma inducer for NK cells. The CG palindrome was also important for activated NK and T cells in their IFN-gamma production, although certain nonpalindromes acted on them. Among the sequences tested, cell activation- or cell lineage-specific sequences were likely; i.e., palindrome ACCGGT and nonpalindrome AACGAT were favored by activated NK cells but not by unactivated NK cells or activated T cells. These results indicate that oligo-DNAs containing CG palindrome act directly on human NK cells and activated T cells to induce IFN-gamma production.     

DNA-templated formation and luminescence of diphenylacetylene dimeric and trimeric complexes

We report on spectral features for two and three diphenylacetylene chromophores aligned in close proximity in aqueous solution by self assembly of attached oligonucleotide arms. Two duplex systems were examined in detail. One was formed by hybridization (Watson-Crick base pairing) of two oligonucleotide 10-mers, each containing the diphenylacetylene insert. The other was generated by self-folding of a 36-mer oligonucleotide containing two diphenylacetylene inserts. The triplex system was obtained by hybridization (Hoogsteen base pairing) of a 16-mer oligonucleotide diphenylacetylene conjugate to the folded 36-mer hairpin. Formation of duplex and triplex entities from these conjugates was demonstrated experimentally by thermal dissociation and spectroscopic studies. The UV and CD spectra for the duplex systems exhibit bands in the 300-350 nm region attributable to exciton coupling between the two chromophores, and the emission spectra show a strong band centered at 410 nm assigned to excimer fluorescence. Addition of the third strand to the hairpin duplex has little effect on the CD spectrum in the 300-350 nm region, but leads to a negative band at short wavelengths characteristic of a triplex and to a strongly enhanced band at 410 nm in the fluorescence spectrum. The third strand alone shows a broad fluorescence band at approximately 345-365 nm, but this band is virtually absent in the triplex system. A model for the triplex system is proposed in which two of the three aligned diphenylacetylenes function as a ground state dimer that on excitation gives rise to the exciton coupling observed in the UV and CD spectra and to the excimer emission observed in the fluorescence spectrum. Excitation of the third chromophore results in enhanced excimer fluorescence, as a consequence of energy transfer from the locally excited singlet of one chromophore to the ground state dimer formed by the other two chromophores.     

荧光能量转移技术在生物学研究中的应用

荧光能量转移(FRET)是指两个携带不同荧光基团的大分子在相互间距离足够近时(10～100A)所发生的能量非放射性地由一个荧光基团向另一个荧光基团转移的现象。结合绿色荧光蛋白的发现,FRET技术可用于检测生物大分子中不同亚基的位置和生物大分子间的相互作用。近年来,FRET技术在生物学研究中的突破性进展是在活体细胞中实时监测生物大分子之间的相互作用。本文就绿色荧光蛋白的发现,FRET技术的原理、研究进展和应用前景作简要综述。     

Strand invasion by DNA-peptide conjugates and peptide nucleic acids.

Peptide nucleic acids (PNAs) and conjugates between oligonucleotides and cationic peptides possess superior potential for strand invasion at complementary sequences. We discovered that oligonucleotide-peptide conjugates and PNAs fall into three classes based on their hybridization efficiency; i) those complementary to inverted repeats within AT-rich region hybridize with highest efficiency; ii) those complementary to areas adjacent to inverted repeats or near AT-rich regions hybridize with moderate efficiency; and iii) those complementary to other regions do not detectably hybridize. The correlations between oligomer chemistry, DNA target sequence, and hybridization efficiency that we report here have important implications for the recognition of duplex DNA.     

Frustration Between Preferred States of Complementary Trinucleotide Repeat DNA Hairpins Anticorrelates with Expansion Disease Propensity

DNA trinucleotide repeat (TRs) expansion beyond a threshold often results in human neurodegenerative diseases. The mechanisms causing expansions remain unknown, although the tendency of TR ssDNA to self-associate into hairpins that slip along their length is widely presumed related. Here we apply single molecule FRET (smFRET) experiments and molecular dynamics simulations to determine conformational stabilities and slipping dynamics for CAG, CTG, GAC and GTC hairpins. Tetraloops are favored in CAG (89%), CTG (89%) and GTC (69%) while GAC favors triloops. We also determined that TTG interrupts near the loop in the CTG hairpin stabilize the hairpin against slipping. The different loop stabilities have implications for intermediate structures that may form when TR-containing duplex DNA opens. Opposing hairpins in the (CAG) ∙ (CTG) duplex would have matched stability whereas opposing hairpins in a (GAC) ∙ (GTC) duplex would have unmatched stability, introducing frustration in the (GAC) ∙ (GTC) opposing hairpins that could encourage their resolution to duplex DNA more rapidly than in (CAG) ∙ (CTG) structures. Given that the CAG and CTG TR can undergo large, disease-related expansion whereas the GAC and GTC sequences do not, these stability differences can inform and constrain models of expansion mechanisms of TR regions.     

Antisense properties of duplex- and triplex-forming PNAs.

The potential of peptide nucleic acids (PNAs) as specific inhibitors of translation has been studied. PNAs with a mixed purine/pyrimidine sequence form duplexes, while homopyrimidine PNAs form (PNA)2/RNA triplexes with complementary sequences on RNA. We show here that neither of these PNA/RNA structures are substrates for RNase H. Translation experiments in cell-free extracts showed that a 15mer duplex-forming PNA blocked translation in a dose-dependent manner when the target was 5'-proximal to the AUG start codon on the RNA, whereas similar 10-, 15- or 20mer PNAs had no effect when targeted towards sequences in the coding region. Triplex-forming 10mer PNAs were efficient and specific antisense agents with a target overlapping the AUG start codon and caused arrest of ribosome elongation with a target positioned in the coding region of the mRNA. Furthermore, translation could be blocked with a 6mer bisPNA or with a clamp PNA, forming partly a triplex, partly a duplex, with its target sequence in the coding region of the mRNA.     

Efficient conjugation and characterization of distamycin-based peptides with selected oligonucleotide stretches

Selected sequences of oligodeoxyribonucleotides (ODNs) have been conjugated efficiently with distamycin-based peptides containing reactive cysteine and oxyamine functionalities at the C-terminus. The conjugation was performed easily within 30-60 min, using individual modified oligonucleotide stretches having sequences of 5'-d(GCTTTTTTCG)-3', 5'-d(GCTATATACG)-3', and 5'-AGCGCGCGCA-3'. Two types of linkages were used for making the covalent connection: (i) a five-membered thiazolidine ring and (ii) an oxime. These distamycin-like polyamide-ODN conjugates were then converted to the corresponding DNA duplexes using complementary oligonucleotide sequences. To elucidate the binding specificity of the distamycin-oligonucleotide conjugates, UV-melting temperature measurements were performed. These studies indicated that the distamycin-ODN conjugate favored binding with the duplex with sequence 5'-d(GCTTTTTTCG)-3' rather than 5'-d(GCTATATACG)-3'. On the other hand, no stabilization of the duplex with sequence 5'-d(AGCGCGCGCA)-3' was observed. UV results also suggest that the thiazolidine and oxime linkages do not significantly influence the process of distamycin binding to the minor groove surface of the DNA duplex. The results obtained from duplex UV-melting studies were further corroborated by a temperature-dependent study of the circular dichroism spectra of the conjugates and a fluorescence displacement titration assay using Hoechst 33258 fluorophore as a competitive binder for the minor groove. All these studies reinforce the fact that the specific stabilization of A/T rich DNA-DNA duplexes by distamycin was preserved upon conjugation with oligonucleotide stretches.     

DNA-directed alkylating ligands as potential antitumor agents: sequence specificity of alkylation by intercalating aniline mustards

The sequence preferences for alkylation of a series of novel parasubstituted aniline mustards linked to the DNA-intercalating chromophore 9-aminoacridine by an alkyl chain of variable length were studied by using procedures analogous to Maxam-Gilbert reactions. The compounds alkylate DNA at both guanine and adenine sites. For mustards linked to the acridine by a short alkyl chain through a para O- or S-link group, 5'-GT sequences are the most preferred sites at which N7-guanine alkylation occurs. For analogues with longer chain lengths, the preference of 5'-GT sequences diminishes in favor of N7-adenine alkylation at the complementary 5'-AC sequence. Magnesium ions are shown to selectively inhibit alkylation at the N7 of adenine (in the major groove) by these compounds but not the alkylation at the N3 of adenine (in the minor groove) by the antitumor antibiotic CC-1065. Effects of chromophore variation were also studied by using aniline mustards linked to quinazoline and sterically hindered tert-butyl-9-aminoacridine chromophores. The results demonstrate that in this series of DNA-directed mustards the noncovalent interactions of the carrier chromophores with DNA significantly modify the sequence selectivity of alkylation by the mustard. Relationships between the DNA alkylation patterns of these compounds and their biological activities are discussed.     

Three-stage refolding/unfolding of the dual-color beta-subunit in R-phycocyanin from Polysiphonia urceolata

The conformational changes during refolding and unfolding of the dual-color beta-subunit in R-phycocyanin (R-PC) were monitored by the spectra, fluorescence anisotropy, and FRET. It was observed that both of the refolding and unfolding of the beta-subunit would undergo a three-stage conformational change, but in a reverse order. During the refolding process, at the first stage, the configuration of the tetrapyrrole chromophores transformed from the cyclohelical to the extended one, suggested by the blue-shifted spectra. At the second stage, recovery of the hydrogen-bond and hydrophobic interaction network fixed the chromophore in a more rigid configuration, suggested by a linear increase in the total fluorescence yield. At the third stage, the increase of the FRET efficiency suggested a protein-framework movement that made the two chromophores closer or/and into a more parallel orientation. The fluorescence anisotropy further confirmed the three-stage model.     

Effect of loop length variation on quadruplex-Watson Crick duplex competition

The effect of loop length on quadruplex stability has been studied when the G-rich strand is present along with its complementary C-rich strand, thereby resulting in competition between quadruplex and duplex structures. Using model sequences with loop lengths varying from T to T5, we carried out extensive FRET to discover the influence of loop length on the quadruplex-Watson Crick duplex competition. The binding data show an increase in the binding affinity of quadruplexes towards their complementary strands upon increasing the loop length. Our kinetic data reveal that unfolding of the quadruplex in presence of a complementary strand involves a contribution from a predominant slow and a small population of fast opening conformer. The contribution from the fast opening conformer increases upon increasing the loop length leading to faster duplex formation. FCS data show an increase in the interconversion between the quadruplex conformers in presence of the complementary strand, which shifts the equilibrium towards the fast opening conformer with an increase in loop length. The relative free-energy difference (Delta DeltaG(o)) between the duplex and quadruplex indicates that an increase in loop length favors duplex formation and out competes the quadruplex.     

Construction and optimization of a family of genetically encoded metabolite sensors by semirational protein engineering

A family of genetically-encoded metabolite sensors has been constructed using bacterial periplasmic binding proteins (PBPs) linearly fused to protein fluorophores. The ligand-induced conformational change in a PBP allosterically regulates the relative distance and orientation of a fluorescence resonance energy transfer (FRET)-compatible protein pair. Ligand binding is transduced into a macroscopic FRET observable, providing a reagent for in vitro and in vivo ligand-measurement and visualization. Sensors with a higher FRET signal change are required to expand the dynamic range and allow visualization of subtle analyte changes under high noise conditions. Various observations suggest that factors other than inter-fluorophore separation contribute to FRET transfer efficiency and the resulting ligand-dependent spectral changes. Empirical and rational protein engineering leads to enhanced allosteric linkage between ligand binding and chromophore rearrangement; modifications predicted to decrease chromophore rotational averaging enhance the signal change, emphasizing the importance of the rotational freedom parameter kappa2 to FRET efficiency. Tighter allosteric linkage of the PBP and the fluorophores by linker truncation or by insertion of chromophores into the binding protein at rationally designed sites gave rise to sensors with improved signal change. High-response sensors were obtained with fluorescent proteins attached to the same binding PBP lobe, suggesting that indirect allosteric regulation during the hinge-bending motion is sufficient to give rise to a FRET response. The optimization of sensors for glucose and glutamate, ligands of great clinical interest, provides a general framework for the manipulation of ligand-dependent allosteric signal transduction mechanisms.     

Location of a contact site between actin and myosin in the three-dimensional structure of the acto-S1 complex

Using fluorescence resonance energy transfer (FRET), we measured distances from chromophores located at or near the actin-binding stretch of amino acids 633-642 of myosin subfragment 1 (S1), to five points in the acto-S1 complex. Specific labeling of this site was achieved by first attaching the desired chromophore to an "antipeptide" that by means of its charge complementarity specifically binds to this segment of S1 [Chaussepied & Morales (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7471] and then cross-linking the fluorescent peptide to the protein. According to this technique, antipeptides containing three different labels, viz., N-dansylaziridine, (iodoacetamido)fluorescein, and monobromobimane, were purified and covalently bound to S1. A second chromophoric group, required for FRET measurements, was selected in such a way as to provide a good spectral overlap with the corresponding peptide chromophore. Cys-707 (SH1) and Cys-697 (SH2) on S1 were modified by using iodoacetamido and maleimido derivatives of rhodamine, 1,N6-ethenoadenosine 5'-diphosphate was trapped at the S1 active site with orthovanadate, Cys-374 on actin was modified with either N-[4-[4-(dimethylamino)phenyl]azo]phenyl]maleimide or N-[(iodoacetyl)-amino]ethyl]-5-naphthylamine-1-sulfonate, and ADP bound to F-actin was exchanged with the fluorescent etheno analogue. By use of excited-state lifetime fluorometry, the following distances from the stretch 633-642 of S1 to other points on S1 or actin have been measured: Cys-707 (S1), 50.3 A; Cys-697 (S1), 49.4 A; active site of S1, greater than or equal to 44 A; nucleotide binding site (actin), 41.1 A; and Cys-374 (actin), approximately 53 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Near-infrared fluorescent oligodeoxyribonucleotide reporters for sensing NF-kappaB DNA interactions in vitro

Two types of reporters for optical sensing of NF-kappaB p50 protein-oligodeoxyribonucleotide (ODN) duplex interactions were designed and compared in vitro. The reporters were based on the effect of fluorescence resonance energy transfer (FRET) between the pair donor Cy5.5 near-infrared (NIR) fluorochrome and either 800CW emitting fluorescence dye acceptor (800CW-Cy), or a nonemitting QSY 21 dye quencher (QSY-Cy). The donor and the acceptor dyes were covalently linked to the complementary oligonucleotides, respectively: Cy dye was conjugated to 3'-thiol, whereas 800CW or QSY21 were conjugated to a hydrophilic internucleoside phosphate amino linker. The reporters were tested initially using recombinant NF-kappaB p50 protein binding assays. Both reporters were binding p50 protein, which protected oligonucleotide duplex from degradation in the presence of exonuclease.The incubation of 800CW-Cy reporter in the presence of control or IL-1beta treated human endothelial cells showed the uptake of the reporter in the cytoplasm and the nucleus. The measurement of NIR fluorescence ratio (i.e. Cy5.5/800CW) showed a partial loss of FRET and the increased Cy5.5 fluorescence in nontreated, control cells. Thus, the specific p50 binding to ODN duplex reporters affected the donor-acceptor fluorochrome pair. NF-kappaB p50 exhibited the protective effect on FRET between NIR fluorochromes linked to the complementary strands of the reporter duplex.     

Quadruplex to Watson-Crick duplex transition of the thrombin binding aptamer: a fluorescence resonance energy transfer study

Thermodynamic parameters of closing up of guanine-rich thrombin binding element, upon binding to K(+) and Na(+) ions to form quadruplexes and opening up of these quadruplexes upon binding to its complementary strand, were investigated. For this purpose, 15mer deoxynucleotide, d(G(2)T(2)G(2)TGTG(2)T(2)G(2)), labeled with 5'-fluorescein and 3'-tetramethylrhodamine was taken and fluorescence resonance energy transfer was monitored as a function of either metal ions or complementary strand concentrations. Equilibrium association constant obtained from FRET studies demonstrates that K(+) ions bind with higher affinity than the Na(+) ions. The enthalpy changes, DeltaH, obtained from temperature dependence of equilibrium association constant studies revealed that formation of quadruplex upon binding of metal ions is primarily enthalpy driven. Binding studies of complementary strand to the quadruplex suggest that opening of a quadruplex in NaCl buffer in presence of the complementary strand is enthalpic as well as entropic driven and can occur easily, whereas opening of the same quadruplex in KCl buffer suffers from enthalpic barrier. Comparison of overall thermodynamic parameters along with kinetics studies indicates that, although quadruplexes cannot efficiently compete with duplex formation at physiological pH, they delay the association of two strands.     

Light activates rotations of bacteriorhodopsin in the purple membrane

To investigate how a photoactivated chromophore drives the proton pump mechanism of bacteriorhodopsin, we have observed how the chromophore rotates during the photocyle. To do this, we examined the dichroism induced in aqueous suspensions of purple membrane fragments by flashes of linearly polarized light. We find that the flash stimulates both the photocycling chromophores and their noncycling neighbors to undergo large (greater than 10 degrees - 20 degrees) rotations within the membrane during the photocycle, and that these two chromophore populations undergo distinctly different sequences of rotations. All these rotations could be eliminated by glutaraldehyde fixation as well as by embedding unfixed fragments in polyacrylamide or agarose gels. Thus, in these immbolizing preparations the chromophore can photocycle without rotating inside a bacteriorhodopsin monomer by more than our detection limit of 2 degrees - 5 degrees. The large rotations we observed in aqueous suspensions of purple membranes were probably due to rotations of entire protein monomers. The process by which a photocycling monomer causes its noncycling neighbors to rotate may help explain the highly cooperative behavior bacteriorhodopsin exhibits when it is aggregated into crystalline arrays of trimers.     

The complete amino-acid sequence of C-phycoerythrin from the cyanobacterium Fremyella diplosiphon

The amino-acid sequences of both subunits of C-phycoerythrin from the cyanobacterium Fremyella diplosiphon have been determined. The alpha-subunit contains 164 amino acid residues, two phycoerythrobilin (PEB) chromophores and has a molecular mass of 18,368 Da (protein: 17,192 Da + 2 PEB, one PEB accounting for 588 Da). The beta-subunit consists of 184 residues, three PEB chromophores and has a molecular mass of 20,931 Da (protein: 19,168 Da and 3 PEB: 1,764 Da). The five PEB chromophores (open chain tetrapyrroles) are covalently bound to six cysteine residues (one of them doubly bound to two cysteine residues). On the alpha-subunit, the first chromophore was found at position 84, homologous to the chromophore binding site of the other biliproteins APC, PC and PEC. The second chromophore, unique for the alpha-subunit of PE, is inserted together with a pentapeptide at position 143 a. On the beta-subunit, a doubly bound chromophore is attached to cysteine residues 50 and 61, similar to the rhodophytan phycoerythrins (B-PE and R-PE). The second and third chromophores were found at positions 84 and 155, homologous to the other biliproteins. A unique peptide insertion of 14 amino acid residues (without chromophore) was found at position 141 a-o in the beta-subunit and probably is located in the three-dimensional model near the additional chromophores of the C-PE alpha- and beta-subunits. Both additional chromophores of the C-PE alpha- and beta-subunit may be located at the periphery of the C-PE-trimer. The amino-acid sequence homology between C-PE alpha- and beta-subunit is 26% and to the alpha- and beta-subunits of C-PC from Mastigocladus laminosus 49% and 48%, respectively.     

Design, biochemical, biophysical and biological properties of cooperative antisense oligonucleotides.

Short oligonucleotides that can bind to adjacent sites on target mRNA sequences are designed and evaluated for their binding affinity and biological activity. Sequence-specific binding of short tandem oligonucleotides is compared with a full-length single oligonucleotide (21mer) that binds to the same target sequence. Two short oligonucleotides that bind without a base separation between their binding sites on the target bind cooperatively, while oligonucleotides that have a one or two base separation between the binding oligonucleotides do not. The binding affinity of the tandem oligonucleotides is improved by extending the ends of the two oligonucleotides with complementary sequences. These extended sequences form a duplex stem when both oligonucleotides bind to the target, resulting in a stable ternary complex. RNase H studies reveal that the cooperative oligonucleotides bind to the target RNA with sequence specificity. A short oligonucleotide (9mer) with one or two mismatches does not bind at the intended site, while longer oligonucleotides (21mers) with one or two mismatches still bind to the same site, as does a perfectly matched 21mer, and evoke RNase H activity. HIV-1 inhibition studies reveal an increase in activity of the cooperative oligonucleotide combinations as the length of the dimerization domain increases.     

Intercalating nucleic acids containing insertions of 1-O-(1-pyrenylmethyl)glycerol: stabilisation of dsDNA and discrimination of DNA over RNA

We have studied hybridisation affinities and fluorescence behaviour of intercalator-modified oligonucleotides. The phosphoramidite of (S)-1-O-(4, 4′-dimethoxytriphenylmethyl)-3-O-(1-pyrenylmethyl)glycerol, an intercalating pseudo-nucleotide (IPN), was synthesised and by standard methods inserted into 7mer and 13mer oligodeoxyribonucleotides (ODNs) to generate intercalating nucleic acids (INAs). INAs showed greatly increased affinity for complementary single-stranded DNA (ssDNA), as determined by a thermal stabilisation of the formed DNA/INA duplex of up to 10.9°C per modification when the IPN was added as a dangling end and up to 6.7°C per modification when the IPN was inserted as a bulge. There was a positive stabilisation effect of the formed DNA/INA duplex on introducing a second IPN in the INA strand, when the two IPNs were separated by at least 1 bp. The effect is more pronounced the larger the separation of the two IPNs. Contrary to the enhanced affinity for ssDNA, the IPNs lower the affinity for complementary single-stranded RNA (ssRNA), giving rise to a difference in melting temperature of up to 25.8°C for two IPN insertions in an RNA/INA duplex when compared with the corresponding DNA/INA duplex. In this way INA is able to discriminate ssDNA over ssRNA with identical sequences. Fluorescence measurements show a stronger interaction of the pyrene moiety with DNA than with RNA, indicating intercalation as the stabilising factor in DNA/INA duplexes.     

Transformation from block-type to graft-type oligonucleotide-glycopolymer conjugates by self-organization with half-sliding complementary oligonucleotides and their lectin recognition

Block-type oligonucleotide-glycopolymer conjugates bearing alpha-mannosides and beta-galactosides were prepared by coupling 5'-thiol-modified oligonucleotides with iodoacetamidated glycopolymers that were synthesized by telomerization. The conjugates minimally affected the DNA conformation and melting behavior of the duplex. Their self-organization via hybridization with the half-sliding complementary oligonucleotides produced graft-type conjugates or macromolecular gapped DNA duplexes grafted with glycopolymers at regular intervals, which was confirmed using size exclusion chromatography and electrophoresis. The binding affinity of block-type and self-organized graft-type conjugates to lectins was investigated using fluorometry. The affinity of the graft-type duplex assembly bearing mannosides to Con A was approximately 2 times stronger than that of block-type single-stranded or double-stranded conjugates with full complementary oligonucleotides. The organization strategy of DNA-glycopolymer conjugates might be useful for constructing novel glyco-clusters and also for developing a new methodology for gene therapy.