Oligodeoxyribonucleotide Sequence Research Articles

Macrophage-targeted nanoparticles mediate synergistic photodynamic therapy and immunotherapy of tuberculosis.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb) that poses a serious global public health threat. Due to the high incidence of adverse reactions associated with conventional treatment regimens, there is an urgent need for better alternative therapies. CpG oligodeoxynucleotides (CpG ODNs) are synthetic oligodeoxyribonucleotide sequences. They can induce a Th1-type immune response by stimulating Toll-like receptors (TLRs) in mammalian immune cells, thus killing Mtb. However, due to the negative charge and easy degradation of CpG ODNs, it is necessary to deliver them into cells using nanomaterials. PCN-224 (hereinafter referred to as PCN), as a metal-organic framework based on zirconium ions and porphyrin ligands, not only has the advantage of high drug loading capacity, but also the porphyrin molecule in it is a type of photosensitizer, which allows these nanocomposites to play a role in photodynamic therapy (PDT) while delivering CpG ODNs. In addition, since Mtb mainly exists in macrophages, targeting anti-TB agents to macrophages is helpful to improve the anti-TB effect. Phosphatidylserine (PS) is a biological membrane phospholipid that is normally found on the inner side of cell membranes in, for example, plant and mammalian cells. When apoptosis occurs, PS can flip from the inner side of the cell membrane to the surface of the cell membrane, displaying a specific "eat-me" signal that can be recognized by specific receptors on macrophages. Therefore, we can use this macrophage-targeting property of PS to construct bio-inspired targeted drug delivery systems. In this study, we constructed PCN-CpG@PS nanocomposites. PCN-CpG@PS, combining PDT and immunotherapy, is designed to target macrophages at the site of a lesion and kill latent Mtb. We physically characterized the nanocomposites and validated their bactericidal ability in vitro and their ability to stimulate the immune system in vivo. The results demonstrated that the targeted nanocomposites have certain in vitro antituberculosis efficacy with good safety.

Minimal sequence requirements for oligodeoxyribonucleotides activating human TLR9.

Synthetic oligodeoxyribonucleotides (ODNs) containing CpG (unmethylated deoxycytidylyl-deoxyguanosine dinucleotide) motifs activate endosomal TLR9. The nucleotide sequence, length, and dimerization properties of ODNs modulate their activation of TLR9. We performed a systematic investigation of the sequence motifs of B-class and C-class phosphodiester ODNs to identify the sequence properties that govern TLR9 activation. ODNs shorter than 21 nt and with the adenosine adjacent to the cytidine-guanosine (CG) dinucleotide motif led to a significant loss of the propensity to activate TLR9. The distance between the stimulatory CpG motifs within the ODN fine-tunes the activation of B cells. The minimal ODNs that activate human TLR9 comprise 2 CG dinucleotides separated by 6-10 nt, where the first CpG motif is preceded by the 5'-thymidine and the elongated poly-thymidine tail at the 3' end of the ODN. The minimal sequence provides insight into the molecular mechanism of TLR9 ligand recognition. On the basis of sequence requirements, we conclude that two binding sites with different affinities for CG are formed in the human TLR9 dimer, with a very stringent binding site interacting with the 5' CpG motif.

Photonic DNA-Chromophore Nanowire Networks: Harnessing Multiple Supramolecular Assembly Modes

Photonic DNA nanostructures are typically prepared by the assembly of multiple sequences of long DNA strands that are conjugated covalently to various dye molecules. Herein we introduce a noncovalent method for the construction of porphyrin-containing DNA nanowires and their networks that uses the programmed assembly of a single, very short, oligodeoxyribonucleotide sequence. Specifically, our strategy exploits a number of supramolecular binding modalities (including DNA base-pairing, metal-ion coordination, and β-cyclodextrin-adamantane derived host-guest interactions) for simultaneous nanowire assembly and porphyrin incorporation. Furthermore, we also show that the resultant DNA-porphyrin assembly can be further functionalized with a complementary "off-the-shelf" DNA binding dye resulting in photonic structures with broadband absorption and energy transfer capabilities.

Sequence-Selective Detection of Double-Stranded DNA Sequences Using Pyrrole–Imidazole Polyamide Microarrays

We describe a microarray format that can detect double-stranded DNA sequences with a high degree of sequence selectivity. Cyclooctyne-derivatized pyrrole-imidazole polyamides were immobilized on azide-modified glass substrates using microcontact printing and a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction. These polyamide-immobilized substrates selectively detected a seven-base-pair binding site incorporated within a double-stranded oligodeoxyribonucleotide sequence even in the presence of an excess of a sequence with a single-base-pair mismatch.

Long Patch Base Excision Repair Proceeds via Coordinated Stimulation of the Multienzyme DNA Repair Complex

Base excision repair, a major repair pathway in mammalian cells, is responsible for correcting DNA base damage and maintaining genomic integrity. Recent reports show that the Rad9-Rad1-Hus1 complex (9-1-1) stimulates enzymes proposed to perform a long patch-base excision repair sub-pathway (LP-BER), including DNA glycosylases, apurinic/apyrimidinic endonuclease 1 (APE1), DNA polymerase beta (pol beta), flap endonuclease 1 (FEN1), and DNA ligase I (LigI). However, 9-1-1 was found to produce minimal stimulation of FEN1 and LigI in the context of a complete reconstitution of LP-BER. We show here that pol beta is a robust stimulator of FEN1 and a moderate stimulator of LigI. Apparently, there is a maximum possible stimulation of these two proteins such that after responding to pol beta or another protein in the repair complex, only a small additional response to 9-1-1 is allowed. The 9-1-1 sliding clamp structure must serve primarily to coordinate enzyme actions rather than enhancing rate. Significantly, stimulation by the polymerase involves interaction of primer terminus-bound pol beta with FEN1 and LigI. This observation provides compelling evidence that the proposed LP-BER pathway is actually employed in cells. Moreover, this pathway has been proposed to function by sequential enzyme actions in a "hit and run" mechanism. Our results imply that this mechanism is still carried out, but in the context of a multienzyme complex that remains structurally intact during the repair process.

Oligonucleotide conjugation to a cell-penetrating (TAT) peptide by Diels–Alder cycloaddition

Modifed oligonucleotides are routinely employed as analytical probes for use in diagnostics, e.g. in the examination of specific RNA sequences for infectious diseases, however, a major limiting factor in oligonucleotide-based diagnostics is poor cellular uptake of naked oligonucleotides. This problem can be overcome by covalent attachment of a so-called 'cell-penetrating peptide' to form an oligonucleotide peptide conjugate. Stepwise solid phase synthesis of such a conjugate is difficult and expensive due to the conflicting chemistries of oligonucleotides and peptides. A simple approach to overcome this is post-synthetic conjugation. Diels-Alder cycloaddition is an attractive methodology for oligonucleotide peptide conjugation; the reaction is fast, chemoselective and the reaction rate is greatly enhanced in aqueous media - ideal conditions for biological moieties. An oligodeoxyribonucleotide sequence has been derivatised with a series of dienes at the 5'-terminus, using a series of unique dienyl-modified phosphoramidites, and investigation into the effect of diene type on the efficiency of conjugation, using Diels-Alder cycloaddition with a maleimido-derivatised cell-penetrating (TAT) peptide, has been performed. This led to the observation that the optimal diene for conjugation was cyclohexadiene, allowing conjugation of oligodeoxyribonucleotides to a cell-penetrating peptide by Diels-Alder cycloaddition for the first time.

Optical Tweezers as a Probe for Oligodeoxyribonucleotide Structuration

The aim of this work is to investigate if the optical tweezers (OT) are suitable as a diagnostic tool for monitoring the oligodeoxyribonucleotide (ODN) structural behavior in solution. Preliminary experiments, performed on the quadruplex formed by the ODN sequence TGGGGT, showed that the OT can be used as a probe for ODN structuration by monitoring the medium viscosity changes associated with ODN folding-unfolding processes.

Betaine, Dimethyl Sulfoxide, and 7-Deaza-dGTP, a Powerful Mixture for Amplification of GC-Rich DNA Sequences

Currently, polymerase chain reaction is the most used technique in many laboratories for either diagnostic or molecular biology purposes. Despite the large number of DNA sequences that can be easily analyzed, some GC-rich sequences are refractory to amplification due to the formation of secondary intramolecular structures. To overcome this problem, several molecules have been described to improve polymerization. Here we show that a combination of three additives--betaine, dimethyl sulfoxide, and 7-deaza-dGTP--was essential to achieve amplification of DNA sequences of three disease genes showing a GC content ranging from 67 to 79%.

Glycomimetics as Decorating Motifs for Oligonucleotides: Solid-Phase Synthesis, Stability, and Hybridization Properties of Carbopeptoid−Oligonucleotide Conjugates

The online solid-phase synthesis of oligonucleotides conjugated at the 3' end with [1-6]-linked oligosaccharide mimics having the O-glycosidic linkages replaced by amide bonds is here described. The assembly of the carbohydrate domain has been carried out by exploiting classical solid phase peptide synthetic protocols, starting from solid supports functionalized with 1-azido sugars, in association with suitably protected 1-azido uronic acids of glucose and lactose, chosen as model addition monomers. After the insertion of a flexible linker, elongation of the oligodeoxyribonucleotide (ODN) chain was performed by standard automated phosphoramidite protocols. 3'-Glycoconjugated 18-mers exhibited an increased enzymatic stability with respect to the same unmodified ODN sequence. UV thermal denaturation experiments showed that the presence of the oligosaccharide tail at the 3' end of the oligonucleotides did not negatively interfere with their duplex formation abilities.

Synthesis of unimolecularly circular G-quadruplexes as prospective molecular probes

Synthesis of unimolecularly circular G-quadruplex has been accomplished for the first time during our investigation on the template basis of G-quadruplex through chemical ligations of guanine-rich linear sequences of oligodeoxyribonucleotides. The uniqueness of this newly designed circularization course is its self-recognition and self-templating on the scale of individual strand of oligodeoxyribonucleotide in which the same linear sequence serves both as a template and as a substrate simultaneously. The results from our exonuclease and DNAse hydrolysis studies confirm that there is indeed absence of open termini within the structure of the identified circular product. Our subsequent investigation on the loop-size effect indicates that the unimolecularly circular G-quadruplex possessing two or more thymine nucleotides within their connecting loops is readily attainable, while the linear sequence with a single thymine nucleotide between guanine tracts is not a proper precursor for our ligation reaction. In addition, conformation dependency of the circularization course as well as the effects of alkali ions, pH values and concentration of potassium ions on the circularization reaction are examined during our investigation. The implication of our current studies and possible application of the obtained unimolecularly circular G-quadruplex in certain biological processes are also discussed in this report.

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.

Hybridization properties of support-bound oligonucleotides: the effect of the site of immobilization on the stability and selectivity of duplex formation.

Four 12-mer oligodeoxyribonucleotide sequences were immobilized to uniformly sized (50 microm) polymer particles through C5-tethered thymine and N(4)-tethered cytosine bases at four different sites in each sequence. The effect of the site of immobilization on the efficiency and selectivity of hybridization of the particle-bound probes was quantified by a sandwich-type assay based on a time-resolved fluorometric measurement of an oligonucleotide probe labeled with a photoluminescent europium(III) chelate directly from the surface of a single particle. Immobilization through a base in the central part of the sequence was observed to destablize the duplex more markedly than tethering through a terminal base. The effect of a one-base mismatch on the duplex stability increased with the increasing distance from the site of immobilization.

Laser-induced photo-cross-linking of cisplatin-modified DNA to HMG-domain proteins.

Laser-induced photo-cross-linking was investigated for DNA, modified with cisplatin at specific sites, bound to structure-specific recognition domains of proteins in the high-mobility group (HMG) class. The efficiency of photo-cross-linking depends on the wavelength and power of the laser, the nature of the protein domain, and the oligodeoxyribonucleotide sequences flanking the platinated site. Introduction of 5-iodouridine at thymine sites of the oligodeoxyribonucleotide as an additional photoreactive group did not increase the photo-cross-linking yield. Formation of platinum-mediated DNA-DNA interstrand cross-linking observed previously upon irradiation with 302 nm light [Kane, S. A., and Lippard, S. J. (1996) Biochemistry 35, 2180-2188] was significantly reduced with laser irradiation. HMG1 domain B is superior to domain A for platinum-mediated photo-cross-linking, a result attributed to the different positioning of the proteins with respect to the platinum adduct and the greater ability of domain B to access photolabilized platinum in the major groove. Studies with proteins containing specifically mutated amino acids, and with DNA probes in which the sequences flanking the platinum cross-link site were varied, suggest that the most effective photo-cross-linking occurs for protein domains bound symmetrically and flexibly to cisplatin-modified DNA. The thermodynamic equilibrium between the protein-platinated DNA complex and its components, revealed in gel electrophoretic mobility shift assays (EMSAs), is significantly shifted to the right upon irreversible photo-cross-linking. Thus, only upon photo-cross-linking can the interaction of cisplatin-DNA 1,3-intrastrand d(GpTpG) or interstrand cross-links with HMG1 domain B protein be detected. Photo-cross-linking is thus an effective tool for investigating the interaction of cisplatin-modified DNA with damage-recognition proteins under heterogeneous conditions such those in cell extracts or living cells.

Expression of individual members of a tRNA1Gly multigene family in vivo follows the same pattern as in vitro

Individual members of a tRNA 1 Gly multigene family from Bombyx mori are transcribed to different levels in vitro in homologous nuclear extracts but their transcription status in vivo is not known. Two sets of tRNA 1 Gly belonging to the extreme groups of highly transcribed and barely transcribed copies have been examined for their expression patterns in vivo in B. mori-derived cell lines following transfection. We have developed a sensitive and reliable method for directly quantifying the transcription levels of transfecting tRNA genes without relying on the biological activity of the transcript. The strategy involved the insertion of synthetic oligodeoxyribonucleotide sequences into the coding region of the transfecting gene and monitoring the transcripts in an RNase protection assay using an antisense probe that clearly distinguished them from the endogenous tRNAs. The oligonucleotide insertion did not significantly affect the transcriptional status of the genes, even though the distance between the A and B boxes was enhanced by 10–15 nt. In vivo also the transcription of tRNA 1 Gly-1 reached very high levels, whereas the transcripts arising from tRNA 1 Gly-6:7 accounted for only 2–5% of the former, closely resembling the transcription patterns in vitro. These individual gene copies having identical coding sequences and consequently the same internal conserved regions, differed only in their flanking sequences which modulate their transcription levels.

Comparison of the base pairing properties of a series of nitroazole nucleobase analogs in the oligodeoxyribonucleotide sequence 5'-d(CGCXAATTYGCG)-3'.

The nucleoside analogs 1-(2'-deoxy-beta-D-ribofuranosyl)- 3-nitropyrrole (9), 1-(2'-deoxy-beta-D-ribofuranosyl)-4-nitropyrazole (10), 1-(2'-deoxy-beta-D-ribofuranosyl)-4-nitroimidazole (11) and 1-(2'-deoxy-beta-D-ribofuranosyl)-5-nitroindole (21) were incorporated into the oligonucleotide 5'-d(CGCXAATTYGCG)-3'in the fourth position from the 5'-end. Procedures for synthesis of two of the nitroazole nucleosides, 10 and 11, were developed for this study. Each of the nitroazoles was converted into a 3'-phosphoramidite for oligonucleotide synthesis by conventional automated protocols. Four oligonucleotides were synthesized for each modified nucleoside in order to obtain duplexes in which each of the four natural bases was placed opposite (position 9) the nitroazole. In order to assess the role of the nitro group on base stacking interaction, sequences were also synthesized in which the fourth base was 1-(2'-deoxy-beta-D-ribofuranosyl)pyrazole. Corresponding sequences containing an abasic site, as well as sequences containing inosine, were synthesized for comparison. Thermal melting studies yielded T m values and thermodynamic parameters. Each nucleoside analog displayed a unique pattern of base pairing preferences. The least discriminating analog was 3-nitropyrrole, for which T m values differed by 5 degrees C and Delta G 25 degrees C ranged from -6.1 to -6.5 kcal/mol. 5-Nitroindole gave duplexes with significantly higher thermal stability, with Tm values varying from 35.0 to 46.5 degrees C and -Delta G 25 degrees C ranging from 7.7 to 8.5 kcal/mol. Deoxyinosine (22), a natural analog which has found extensive use as a universal nucleoside, is far less non-discriminating than any of the nitroazole derivatives. Tm values ranged from 35.4 degrees C when paired with G to 62.3 degrees C when paired with C. The significance of the nitro substituent was determined by comparison of the base pairing properties of a simple azole nucleoside, 1-(2'-deoxy-beta-D-ribofuranosyl)pyrazole (12). The pyrazole-containing sequences melt at 10-20 degrees C lower than the corresponding nitropyrazole-containing sequences. On average, the pyrazole-containing sequences were equivalent in stability (average Delta G = -4.8 kcal/mol) to the sequences containing an abasic site (average Delta G = -4.7 kcal/mol).

Acetal Oligonucleotide Conjugates in Antisense Strategy

To enhance the cellular uptake for antisense oligonucleotides or analogues a lipophilic steroid moiety was linked to the 5′-O- or 2′-O-position of appropriate protected thymidine or uridine simply by acid catalysed reaction with cholesterylvinylether. The corresponding cholesteryl-acetals were derivatized to the 3′-O- or 5′-O-phosphoramidites and then introduced as 5′-end terminating agents or incorporated within the sequence of oligodeoxyribonucleotides up to a chain length of 18 bases. 3′ -end linkage was achieved by using the corresponding 2′-O-cholesteryluridine building unit, tethered via a 3′-O-/5′-O-succinate-bridge to polystyrene as solid support.

Rainbow™ Universal CPG: A Versatile Solid Support for Oligonucleotide Synthesis

We have developed a universal solid support, termed Rainbow Universal CPG, for use in automated oligonucleotide synthesis. The universal solid support allows any oligodeoxyribonucleotide sequence to be synthesized from a single type of controlled pore glass (CPG) support. Deprotection of oligodeoxyribonucleotides was optimized using 0.5 M LiCl in concentrated ammonium hydroxide. PCR experiments using three different sets of primers proved that the 3'-hydroxyl function of oligodeoxyribonucleotides synthesized from Rainbow Universal CPG was retained. This universal solid support shows promise for replacing the standard nucleoside CPG supports.

Applications of delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry to oligonucleotide analysis.

A delayed ion extraction technique is shown to dramatically improve mass resolution and the overall quality of matrix-assisted laser desorption ionization (MALDI) mass spectra of oligonucleotides. Isotope limited mass resolution was obtained on samples up to 10-kDa molecular mass in linear mode, and as high as 7500 mass resolution (defined at half peak height) was observed in reflector mode. This performance is as good as that achieved to date for peptides and proteins. Applications included the detection of oxidized byproducts of phosphorothioate DNA and separation of components differing only by 15 Da at 9.5-kDa molecular mass. In addition to single components, complex mixtures could also be analyzed at greatly improved performance over conventional MALDI. An example is shown for sequence verification of an oligonucleotide of 31 bases in length by analyzing the failure products. Mass accuracy was adequate to verify sequences of oligodeoxyribonucleotides up to 9500-Da molecular mass. Fast fragmentation taking place between the ionizing pulse and the extraction pulse is demonstrated to be a sequencing tool for small oligonucleotides. By proper selection of matrix material, wavelength, and irradiance, fast fragmentation can be promoted efficiently. Fragment ions tend to form from cleavage of phosphodiester bonds, as previously observed in infrared MALDI.

Solution structures of 5-fluorouracil-substituted DNA and RNA decamer duplexes.

The structures in solution of eight oligonucleotide duplexes each containing either zero, one, or two 5-fluorodeoxyuridine (FdUrd) or 5-fluorouridine (FUrd) nucleosides were determined by the combined use of NMR spectroscopy, restrained molecular dynamics, and full relaxation matrix refinement to determine how FdUrd and FUrd substitution affects the structure of duplex DNA and RNA and to establish whether structural differences due to FdUrd and FUrd substitution in nucleic acids may be responsible, in part, for the biological effects of the anticancer drug 5-fluorouracil (FUra). The nucleic acid directed effects of FUra include induction of single-strand breaks in duplex DNA and altered processing of pre-mRNA and rRNA. Four self-complementary oligodeoxyribonucleotide sequences were prepared and studied as duplexes in aqueous solution: (5' dGCGAAUUCGC)2, (5' dGCGAAUFCGC)2, (5' dGCGAAFUCGC)2, and (5' dGCGAAFFCGC)2. The corresponding oligoribonucleotide sequences (5' rGCGAAUUCGC)2, (5' rGCGAAUFCGC)2, (5' rGCGAAFUCGC)2, and (5' rGCGAAFFCGC)2 were also prepared and studied. The helical parameters for the structures of these eight duplexes were analyzed to determine how substitution of FdUrd and FUrd affects the three-dimensional structures of duplex DNA and RNA. FdUrd substitution affects the base roll angle at the site of FdUrd substitution, causing the helical axis of FdUrd-substituted DNA duplexes to be bent compared to the nonsubstituted duplex. A-FUrd base pairs show substantial RMS deviations from A-Urd base pairs in all three of the RNA duplexes substituted with FUrd. Bending of the helical axis due to FdUrd substitution may contribute to the occurrence of single-strand breaks in duplex DNA while the altered structures of A-FUrd base pairs may affect RNA-RNA and RNA-protein recognition.

BCR-ABL Antisense Oligodeoxyribonucleotides Suppress the Growth of Leukemic and Normal Hematopoietic Cells by a Sequence-Specific But Nonantisense Mechanism

We have examined the effect of BCR/ABL junctional antisense phosphodiester oligodeoxyribonucleotides (ODNs) on BV173 and other chronic myeloid leukemia (CML) cell lines. Various control ODNs were used to understand the mechanism of the observed antiproliferative effect. Not only the antisense ODNs but also several control ODNs inhibit the proliferation of the leukemic cell lines. All the ODNs that inhibit the cell proliferation share a TAT consensus sequence at their 3' end. A 1-base mismatch within this consensus sequence abolishes the antiproliferative effect. Mismatches of several bases at any other position within the sequence of the active ODNs do not suppress the observed effect. Similar experiments on normal or CML CD34+ cell fraction led to the same observations. We conclude that the antiproliferative effect of the phosphodiester BCR/ABL antisense ODNs cannot be attributed to an antisense mechanism but rather to a nonelucidated effect of a 3' terminal TAT sequence. This effect is not CML specific.