Synthesis of an oligodeoxyribonucleotide adduct of mitomycin C by the postoligomerization method via a triamino mitosene

The cancer chemotherapeutic agent mitomycin C (MC) alkylates and cross-links DNA monofunctionally and bifunctionally in vivo and in vitro, forming six major MC-deoxyguanosine adducts of known structures. The synthesis of one of the monoadducts (8) by the postoligomerization method was accomplished both on the nucleoside and oligonucleotide levels, the latter resulting in the site-specific placement of 8 in a 12-mer oligodeoxyribonucleotide 26. This is the first application of this method to the synthesis of a DNA adduct of a complex natural product. Preparation of the requisite selectively protected triaminomitosenes 14 and 24 commenced with removal of the 10-carbamoyl group from MC, followed by reductive conversion to 10-decarbamoyl-2,7-diaminomitosene 10. This substance was transformed to 14 or 24 in several steps. Both were successfully coupled to the 2-fluoro-O(6)-(2-trimethylsilylethyl)deoxyinosine residue of the 12-mer oligonucleotide. The N(2)-phenylacetyl protecting group of 14 after its coupling to the 12-mer oligonucleotide could not be removed by penicillinamidase as expected. Nevertheless, the Teoc protecting group of 24 after coupling to the 12-mer oligonucleotide was removed by treatment with ZnBr2 to give the adducted oligonucleotide 26. However, phenylacetyl group removal was successful on the nucleoside-level synthesis of adduct 8. Proof of the structure of the synthetic nucleoside adduct included HPLC coelution and identical spectral properties with a natural sample, and (1)H NMR. Structure proof of the adducted oligonucleotide 26 was provided by enzymatic digestion to nucleosides and authentic adduct 8, as well as MS and MS/MS analysis.     

Aptamer-based electrochemical sensors that are not based on the target binding-induced conformational change of aptamers

This study describes a new kind of aptamer-based electrochemical sensor that is not based on the target binding-induced conformational change of the aptamers by using a 15-mer thrombin-binding aptamer (5'-GGTTGGTGTGGTTGG-3') as the model oligonucleotide. The sensors are developed by first self-assembling the aptamer (i.e. a thrombin-binding aptamer) onto an Au electrode and then hybridizing the assembled aptamer with a ferrocene (Fc)-labeled short aptamer-complementary DNA oligonucleotide to form an electroactive double-stranded DNA (ds-DNA) oligonucleotide onto the Au electrode. The binding of the target (i.e. thrombin) towards the aptamer essentially destroys the Watson-Crick helix structure of the ds-DNA oligonucleotide assembled onto the electrode and leads to the dissociation of the Fc-labeled short complementary DNA oligonucleotide from the electrode surface to the solution, resulting in a decrease in the current signal obtained at the electrode, which can be used for the determination of the target. With the thrombin-binding aptamer as the model oligonucleotide, the current decrease obtained with the aptamer-based electrochemical sensors is linear with the concentration of thrombin within the concentration range from 0 to 10 nM (DeltaI/nA = 6.7C(thrombin)/nM + 2.8, gamma = 0.975). Unlike most kinds of existing aptamer-based electrochemical sensor, the electrochemical aptasensors demonstrated here are not based on the conformational change of the aptamers induced by the specific target binding. Moreover, the aptasensors are essentially label-free and are very responsive toward the targets. This study may pave a facile and general way to the development of aptamer-based electrochemical sensors.     

Increasing the Sensitivity and Single‐Base Mismatch Selectivity of the Molecular Beacon Using Graphene Oxide as the “Nanoquencher”

Here, we report a novel, highly sensitive, selective and economical molecular beacon using graphene oxide as the “nanoquencher”. This novel molecular beacon system contains a hairpin‐structured fluorophore‐labeled oligonucleotide and a graphene oxide sheet. The strong interaction between hairpin‐structured oligonucleotide and graphene oxide keep them in close proximity, facilitating the fluorescence quenching of the fluorophore by graphene oxide. In the presence of a complementary target DNA, the binding between hairpin‐structured oligonucleotide and target DNA will disturb the interaction between hairpin‐structured oligonucleotide and graphene oxide, and release the oligonucleotide from graphene oxide, resulting in restoration of fluorophore fluorescence. In the present study, we show that this novel graphene oxide quenched molecular beacon can be used to detect target DNA with higher sensitivity and single‐base mismatch selectivity compared to the conventional molecular beacon.     

A mechanistic study of the electron capture dissociation of oligonucleotides

Electron capture dissociation (ECD) of a series of custom-synthesized oligonucleotide pentamers was performed in a Fourier-transform mass spectrometer with a conventional filament-type electron gun. Dissociation of oligonucleotide ions by electron capture generates primarily w/d-type and z/a-type ions with and without the loss of a nucleobase fragment ions. Minor yields of radical [z/a + H]. fragment ions were also observed in many cases. It is interesting to note that some nucleoside-like fragment ions and protonated nucleobase ions (except thymine-related nucleobases and nucleoside-like fragments) were observed in most ECD spectra. The formation of these low-mass fragment ions was tentatively attributed to the secondary fragmentation of the radical [z + H]. fragment ions. From the ECD tandem mass spectra of a series of C/T based binary oligonucleotide ions, including d(CTCTC), d(CTTTC), d(TCCCT), d(CCCCT), and d(TCCCC), it was clearly demonstrated that the formation of many sequence ions was sensitive to the position of cytosine (or the position of charge carrier). The findings of this work support a notion that the ECD of protonated oligonucleotide molecules is charge-directed with the electron being captured by the protonated nucleobase.     

Use of an amino-silica column for the high-performance liquid chromatographic analysis of synthetic oligodeoxy-nucleotides.

The use of an amino-silica column in the chromatographic analysis of synthetic oligodeoxyribonucleotides and their derivatives from different stages of oligonucleotide synthesis has been investigated. By eluting with 0.10 M potassium phosphate solution of pH 3.30, the nucleotide composition of oligonucleotides can be established within 15 min. In a linear gradient of phosphate buffer (0.10-0.75 M) at neutral pH, the separation of oligonucleotides by length and in an acidic medium pH 3.30-4.30) by composition is possible; the oligonucleotides may be in the free form or modified by the various protecting groups used in synthetic oligonucleotide chemistry. The analysis of some reaction mixtures from different stages of oligonucleotide synthesis and of a number of synthetic oligodeoxyribonucleotides and their derivatives has been performed.     

寡聚核苷酸/单链阳离子表面活性剂囊泡与沉淀共存(英文)

DNA(包括寡聚核苷酸)和阳离子表面活性剂可形成难溶复合物.本文通过浊度测试和透射电子显微镜观察,发现单链阳离子表面活性剂可以诱使寡聚核苷酸/单链阳离子表面活性剂沉淀转变成为寡聚核苷酸/单链阳离子表面活性剂囊泡,且寡聚核苷酸/单链阳离子表面活性剂囊泡可以与寡聚核苷酸/单链阳离子表面活性剂沉淀共存.在寡聚核苷酸/单链阳离子表面活性剂沉淀向囊泡的转变过程中,表面活性剂和沉淀之间的疏水作用力发挥了重要作用.此外,当体系温度达到寡聚核苷酸开始融解的温度后,寡聚核苷酸/单链阳离子表面活性剂体系更容易形成囊泡.因此,寡聚核苷酸的链越伸展,越易于寡聚核苷酸/单链阳离子表面活性剂囊泡的生成.据我们所知,有关寡聚核苷酸/阳离子表面活性剂囊泡的报道尚不多见.因此,考虑到DNA(包括寡聚核苷酸)/两亲分子体系在医学、生物学、药学和化学中的重要性,该研究应该有助于我们进一步了解该体系并对其进行更合理有效的应用.     

Tandem oligonucleotide synthesis on solid-phase supports for the production of multiple oligonucleotides

More than one oligonucleotide can be synthesized at a time by linking multiple oligonucleotides end-to-end in a tandem manner on the surface of a solid-phase support. The 5'-terminal hydroxyl position of one oligonucleotide serves as the starting point for the next oligonucleotide synthesis. The two oligonucleotides are linked via a cleavable 3'-O-hydroquinone-O,O'-diacetic acid linker arm (Q-linker). The Q-linker is rapidly and efficiently coupled to the 5'-OH position of immobilized oligonucleotides using HATU, HBTU, or HCTU in the presence of 1 equiv of DMAP. This protocol avoids introduction of phosphate linkages on either the 3'- or 5'-end of oligonucleotides. A single NH(4)OH cleavage step can simultaneously release the products from the surface of the support and each other to produce free 5'- and 3'-hydroxyl termini. Selective cleavage of one oligonucleotide out of two sequences has also been accomplished via a combination of succinyl and Q-linker linker arms. Tandem synthesis of multiple oligonucleotides is useful for producing sets of primers for PCR, DNA sequencing, and other diagnostic applications as well as double-stranded oligonucleotides. Tandem synthesis of the same sequence multiple times increases the yield of material from any single synthesis column for maximum economy in large-scale synthesis. This method can also be combined with reusable solid-phase supports to further reduce the cost of oligonucleotide production.     

DNAzyme-Catalyzed Site-Specific N-Acylation of DNA Oligonucleotide Nucleobases

We used in vitro selection to identify DNAzymes that acylate the exocyclic nucleobase amines of cytidine, guanosine, and adenosine in DNA oligonucleotides. The acyl donor was the 2,3,5,6-tetrafluorophenyl ester (TFPE) of a 5′-carboxyl oligonucleotide. Yields are as high as >95 % in 6 h. Several of the N-acylation DNAzymes are catalytically active with RNA rather than DNA oligonucleotide substrates, and eight of nine DNAzymes for modifying C are site-specific (>95 %) for one particular substrate nucleotide. These findings expand the catalytic ability of DNA to include site-specific N-acylation of oligonucleotide nucleobases. Future efforts will investigate the DNA and RNA substrate sequence generality of DNAzymes for oligonucleotide nucleobase N-acylation, toward a universal approach for site-specific oligonucleotide modification.     

Effects of Acid and Base on the Inductive Efficiency of Oligonucleotide on the Vesicle Formation from Single-Chained Cationic Surfactant

Recently, we reported for the first time that oligonucleotide could induce single‐chained cationic surfactant molecules to aggregate into vesicles and the facilitative efficiency of oligonucleotide on vesicle formation was dependent on its size and sequence. In the present paper, we will continue to study the effects of acid and base on the facilitative efficiency of oligonucleotide on vesicle formation. It is found that proton ions show little effect on the facilitative efficiency while hydroxide ions make it decreased. Moreover, the percentage of oligonucleotide involved in vesicle formation in basic solution is much lower than that in acidic solution (which is almost equal to that in water). Since the structures and properties of DNA/amphiphile complex are very important for its application as nonviral gene carrier, this study may provide some helpful information for gene therapy.     

Promiscuous Watson-Crick cross-pairing within the family of pentopyranosyl (4'-->2') oligonucleotides

[formula: see text] The D-beta-ribo, D-beta-xylo, L-alpha-lyxo, and L-alpha-arabino members of the pentopyranosyl (4'-->2') oligonucleotide family show efficient intersystem cross-pairing among each other. This family of configurationally isomeric and conformationally well-defined pairing systems offers an opportunity to study structural factors that determine cross-communication between informational oligonucleotide systems of different backbone structure.     

应用微阵列芯片检测与2型糖尿病相关的单核苷酸多态性

基于三维(3D)寡核苷酸微阵列芯片的荧光检测法, 研制了一种用于筛选能检测2型糖尿病的特定寡核苷酸探针. 使用第4代(G4)聚(酰胺-胺)(PAMAM)树枝状大分子修饰的载玻片为基底, 以氨基修饰的寡核苷酸为固定探针构建3D寡核苷酸微阵列芯片. 采用荧光化合物Cy5修饰的寡核苷酸为检测探针获得荧光信号. 以2型糖尿病易感基因TCF7L2的rs7903146位点为研究对象, 通过对含有16种(8对)寡核苷酸的寡核苷酸文库的筛选, 获得了1对能用于2型糖尿病检测的寡核苷酸探针. 通过单核苷酸多态性和等位基因分析证明, 该寡核苷酸探针对靶标寡核苷酸检测具有高特异性, 并能准确检测低至2%的等位基因频率.     

Sensitized photomodification of oligonucleotide-binding proteins displayed on the eucaryotic cell surface

Interactions of double-stranded nucleic acids with cell surface proteins, which are involved in binding and transport of extracellular nucleic acids, were studied by the photoaffinity modification with a binary system of oligonucleotide conjugates. The photoreactive double-stranded complex involved an oligonucleotide template and two complementary to adjacent sequences oligonucleotide conjugates. One conjugate contained a photoreagent, viz., 4-azido-2,3,5,6-tetrafluorobenzaldehyde N-(3-aminopropionyl)hydrazone, at the terminus located in proximity to the terminus of another conjugate containing the sensitizer, viz., 9-aminomethylanthracene. Binding of photoreagent and the sensitizer to a single-stranded template yields the photoreactive center. Upon irradiation with visible light (400—580 nm), this photoreactive double-stranded complex forms covalent cross-linkages with oligonucleotide-binding surface proteins of eucaryotic SPEV cells.     

Maleimide-dimethylfuran exo adducts: effective maleimide protection in the synthesis of oligonucleotide conjugates

The reaction of maleimide-containing compounds with 2,5-dimethylfuran gives a mixture of exo and endo isomers from which the exo cycloadduct can be easily isolated taking advantage of its stability in concentrated aqueous ammonia. Bifunctional compounds incorporating a dimethylfuran-protected maleimide (exo adduct) have been attached to resin-linked oligonucleotide chains. Removal of protecting groups masking oligonucleotide functionalities followed by retro-Diels-Alder maleimide deprotection affords maleimido-oligonucleotides suitable for conjugation, as assessed by their reaction with different thiols.     

New phosphoramidite derivatives for the preparation of oligonucleotides containing a hydrazide group in any specified position of the oligonucleotide chain

A new versatile method for the preparation of oligonucleotides containing hydrazide groups in any position of the oligonucleotide chain by standard phosphoramidite automated oligonucleotide synthesis is proposed. The method is based on the use of a series of new modified components for oligonucleotide synthesis. An original protecting group for the hydrazide group is proposed. The presence of the hydrazide group in the obtained oligonucleotides and its high reactivity were demonstrated by the reaction with 4-methoxybenzaldehyde in solution. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 11, pp. 2585–2595, November, 2005.     

Anti-fouling characteristics of surface-confined oligonucleotide strands bioconjugated on streptavidin platforms in the presence of nanomaterials

This work describes our studies on the molecular design of interfacial architectures suitable for DNA sensing which could resist non-specific binding of nanomaterials commonly used as labels for amplifying biorecognition events. We observed that the non-specific binding of bio-nanomaterials to surface-confined oligonucleotide strands is highly dependent on the characteristics of the interfacial architecture. Thiolated double stranded oligonucleotide arrays assembled on Au surfaces evidence significant fouling in the presence of nanoparticles (NPs) at the nanomolar level. The non-specific interaction between the oligonucleotide strands and the nanomaterials can be sensitively minimized by introducing streptavidin (SAv) as an underlayer conjugated to the DNA arrays. The role of the SAv layer was attributed to the significant hydrophilic repulsion between the SAv-modified surface and the nanomaterials in close proximity to the interface, thus conferring outstanding anti-fouling characteristics to the interfacial architecture. These results provide a simple and straightforward strategy to overcome the limitations introduced by the non-specific binding of labels to achieve reliable detection of DNA-based biorecognition events.     

Enzymatic nanolithography of a self-assembled oligonucleotide monolayer on gold

This paper describes a simple strategy to biochemically manipulate a surface at the nanoscale by enzyme dip-pen nanolithography using an endonuclease (DNase I) that is directly patterned on a self-assembled monolayer presenting a terminal oligonucleotide. Physisorbed nanopatterns of DNase I carried out nanoscale enzymology at the surface creating oligonucleotide patterns with the fidelity of the patterned enzyme because of the affinity of the enzyme for the immobilized, oligonucleotide substrate.     

Electrochemical response of oligonucleotides on carbon paste electrode

Electrochemical response of synthetic oligonucleotides with different DNA bases sequences was investigated to find relationships between a chain composition and a signal. All DNA mononucleotides present electroactivity at a carbon paste electrode yielding anodic peaks at potentials: 1.00 (GMP), 1.28 (AMP), 1.47 (TMP) and 1.53 V (CMP). Also 15-mer homooligonucleotides show respective anodic peaks. Electrochemical response of 15- and 19-mer oligonucleotides consisting of all four DNA bases in different amounts was determined by the composition of oligonucleotide chain. When the contribution of different bases in oligonucleotide was balanced two anodic peaks were obtained that can be attributed to guanine and adenine moieties. Thymine residue is shown as a separate peak in voltammogram when its content in oligonucleotide chain is close to 50% of the total number of bases. Cytosine also yields a peak at its significant contribution in oligonucleotide chain and both pyrimidinic moieties produce catalytic waves easier when one of them is dominating or when only one pyrimidine derivative is present in a chain. Guanine is the easiest oxidized base and it produces a peak even at its minimal contribution (one guanine residue in 19-mer oligonucleotide). Guanine peak potential is dependent on oligonucleotide concentration and oligonucleotide composition. The lowest oligonucleotide concentration detected by guanine peak was 12.5 nM whereas detected by thymine peak was 90 nM.     

New solid support for the synthesis of 3'-oligonucleotide conjugates through glyoxylic oxime bond formation

A novel solid support 1 was synthesized to incorporate glyoxylic aldehyde functionality at the oligonucleotide 3'-terminus. 6-mer and 11-mer oligonucleotide sequences containing 3'-glyoxylic aldehyde functionality were prepared by using this support. These modified oligonucleotides were coupled to reporters containing an aminooxy group to prepare oligonucleotide 3'-conjugates through glyoxylic oxime bond formation. The hydrolytic stability of a glyoxylic oxime linkage was also investigated. [reaction: see text].     

Amperometric detection of DNA hybridization on a gold surface depends on the orientation of oligonucleotide chains

We tested the possibility of amperometric detection of DNA hybridization on a gold surface influenced by the immobilization of oligonucleotide giving different orientations of single stranded DNA relative to the gold surface. The DNA sensor was fabricated by chemisorption of 18-mer oligonucleotide modified by a phosphorothioate group either at its 3' or both 3' and 5' terminal. After immobilization of oligonucleotide to the gold support, the sensor was immersed in 11-mercaptoundecanoic acid (MUA) solution. Further chemisorption of MUA resulted in approximately 10-fold increase of resistance of the organic layer. Addition of complementary oligonucleotide resulted in an increase of conductivity for DNA sensor oriented perpendicular to the gold support (DNA with one thiol group), while the conductance decreased for DNA sensor with single stranded DNA oriented parallel to the gold support (with DNA modified by thiol groups at both 3' and 5' terminals). Addition of non-complementary chain resulted a slight decrease or no change of sensor conductivity. The hybridization process at both types of DNA orientations is not cooperative and can be described by Langmuir isotherms. The hybridization event on gold support has been confirmed by mass detection using the quartz crystal microbalance technique.     

Electrochemiluminescent detection of the hybridization of oligonucleotides using an electrode modified with nanocomposite of carbon nanotubes and gold nanoparticles

We report on an electrochemiluminescent (ECL) sensing technique for the detection of the hybridization between oligonucleotides. A glassy carbon electrode was first functionalized with a composite prepared from gold nanoparticles and carbon nanotubes, and a sensor was then constructed by immobilizing the probing oligonucleotide. The ECL of luminol acts as the sensing signal. It is quenched, to a different degree, by the hybridized double strands of the oligonucleotide depending on the match status. The slope of the ECL response as a function of the status of hybridization drops with increasingly matched hybridization. The response is attributed to the interaction between luminol and the strands of oligomers, and also related to the reduction of reactive oxygen species.