Synthesis of aminoglycoside-modified oligonucleotides

[structure: see text] To study the structural requirements of aminoglycoside binding to nucleic acids, compound 1-an analogue of the naturally occurring nucleoside J-was synthesized. When incorporated into oligodeoxynucleotides, 1 leads to thermal stabilization of the resulting duplexes. The increase in pairing affinity is stronger with complementary RNA than with DNA.     

DNA-selective hybridization and dual strand invasion of short double-stranded DNA using pyren-1-ylcarbonyl-functionalized 4'-C-piperazinomethyl-DNA

Incorporation of a novel pyren-1-ylcarbonyl-functionalized 4'-C-piperazinomethyl-DNA monomer into oligodeoxynucleotides leads to increased thermal stability of duplexes with DNA complements but reduced thermal stability of duplexes with RNA complements. This DNA-selective hybridization is explored for recognition of double-stranded DNA by a novel dual strand invasion approach.     

Synthesis and characterization of fluorenone-, anthraquinone-, and phenothiazine-labeled oligodeoxynucleotides: 5'-probes for DNA redox chemistry

A facile and automated procedure for the synthesis of oligodeoxynucleotides possessing derivatives of 9-fluoreneone, 9,10-anthraquinone, and phenothiazine is described. The phosphoramidite approach is used to attach these redox and spectroscopic probes to the 5'-terminus of oligodeoxynucleotides in high yield (>95%). Thermal denaturation studies of labeled oligodeoxynucleotides show a slight enhancement in duplex stability relative to the unlabeled control, and circular dichroism spectra confirm B-form helical DNA structure in solution.     

Nucleic acid secondary structures containing the double-headed nucleoside 5'(S)-C-(2-(thymin-1-yl)ethyl)thymidine

Oligodeoxynucleotides containing the double-headed nucleoside 5'(S)-C-(2-(thymin-1-yl)ethyl)thymidine were prepared by standard solid phase synthesis. The synthetic building block for incorporating the double-headed moiety was prepared from thymidine, which was stereoselectively converted to a protected 5'(S)-C-hydroxyethyl derivative and used to alkylate the additional thymine by a Mitsunobu reaction. The oligodeoxynucleotides were studied in different nucleic acid secondary structures: duplexes, bulged duplexes, three-way junctions and artificial DNA zipper motifs. The thermal stability of these complexes was studied, demonstrating an almost uniform thermal penalty of incorporating one double-headed nucleoside moiety into a duplex or a bulged duplex, comparable to the effects of the previously reported double-headed nucleoside 5'(S)-C-(thymin-1-yl)methylthymidine. The additional base showed only very small effects when incorporated into DNA or RNA three-way junctions. The various DNA zipper arrangements indicated that extending the linker from methylene to ethylene almost completely removed the selective minor groove base-base stacking interactions observed for the methylene linker in a (-3)-zipper, whereas interactions, although somewhat smaller, were observed for the ethylene linker in a (-4)-zipper motif.     

Synthesis and properties of trans-3',4'-bridged nucleic acids having typical S-type sugar conformation

The synthesis of nucleoside analogues with a conformationally restricted sugar moiety is of great interest. The present research describes the synthesis of BNA (bridged nucleic acid) monomers 1 and 2 bearing a 4,7-dioxabicyclo[4.3.0]nonane skeleton and a methoxy group at the C2' position. Conformational analysis showed that the sugar moiety of these monomers is restricted in a typical S-type conformation. It was difficult to synthesize the phosphoramidite derivative of the ribo-type monomer 1, while the phosphoramidite of the arabino-type monomer 2 was successfully prepared and incorporated into oligodeoxynucleotides (ODNs). The hybridization ability of the obtained ODN derivatives containing 2 with complementary strands was evaluated by melting temperature (T(m)) measurements. As a result, the ODN derivatives hybridized with DNA and RNA complements in a sequence-selective manner, though the stability of the duplexes was lower than that of the corresponding natural DNA/DNA or DNA/RNA duplex.     

Synthesis and Properties of Carbocyclic 5′- Nor Oligodeoxynucleotides as Potential Antisense Molecules

We have synthesized carbocyclic 5'-nor oligodeoxynucleotides with a shortened overall chain length lacking the 5'-methylene. Their hybridization properties with DNA and RNA were investigated by UV and CD melting curves. These oligomers formed unstable duplexes with DNA, but formed stable duplexes with RNA selectively. In addition, these oligomers were very stable against nucleases.     

Sequence‐Dependent Duplex Stabilization upon Formation of a Metal‐Mediated Base Pair

An artificial nucleoside surrogate with 1H‐imidazo[4,5‐f][1,10]phenanthroline ( P ) acting as an aglycone has been introduced into DNA oligonucleotide duplexes. This nucleoside surrogate can act as a bidentate ligand, and so is useful in the context of metal‐mediated base pairs. Several duplexes involving a hetero base pair with an imidazole nucleoside have been investigated. The stability of DNA duplexes incorporating the respective Ag^I‐mediated base pairs strongly depends on the sequence context. Quantum mechanical/molecular mechanical (QM/MM) calculations have been performed in order to gain insight into the factors determining this sequence dependence. The results indicated that, in addition to the stabilizing effect that results from the formation of coordinative bonds, destabilizing effects may occur when the artificial base pair does not fit optimally into the surrounding B‐DNA duplex.     

Synthesis and modelling of DNA junction and minor groove zipper motifs incorporating the double-headed nucleoside 5'(S)-C-(thymine-1-ylmethyl)thymidine

A nucleoside with two nucleobases, a so-called double-headed nucleoside, 5'(S)-C-(thymine-1-ylmethyl)thymidine 3, is synthesised and incorporated into oligonucleotides. The additional nucleobase is hereby positioned in the minor groove of the duplexes, which are formed with complementary DNA and RNA-sequences. Slight thermal destabilisation of these duplexes as compared to unmodified duplexes is observed. With other target sequences forming bulged duplexes or three-way junctions, no additional influence of the additional base on the thermal stability is observed. On the other hand, a base-base stacking interaction and subsequent stabilisation is observed when two double-headed nucleotide moieties are positioned in two complementary DNA-sequences forming a DNA-zipper motif.     

Low Tm DNA duplexes observed by cold-spray ionization mass spectrometry

Double-stranded oligodeoxynucleotides (6- to 14-mer) were observed by using CSI-MS. This method made it possible to observe very unstable species such as low Tm DNA duplexes which can not be detectable by conventional ESI-MS.     

Synthesis and RNA-selective hybridization of α-l-ribo- and β-d-lyxo-configured oligonucleotides

Three α-l-ribofuranosyl analogues of RNA nucleotides (α-l-RNA analogues) have been synthesized and incorporated into oligonucleotides using the phosphoramide approach on an automated DNA synthesizer. The 4′-C-hydroxymethyl-α-l-ribofuranosyl thymine monomer was furthermore synthesized. Relative to the unmodified duplexes, incorporation of a single α-l-RNA monomer into a DNA strand leads to reduced thermal stability of duplexes with DNA complements but unchanged thermal stability of duplexes with RNA complements, whereas incorporation of more than one α-l-RNA monomer lead to moderately decreased thermal stability also of duplexes with RNA complements. Efficient hybridization with an RNA complement and no melting transition with a DNA complement were observed with stereoregular chimeric oligonucleotides composed of a mixture of α-l-RNA and affinity enhancing α-l-LNA monomers (α-l-ribo-configured locked nucleic acid). Furthermore, duplexes formed between oligodeoxynucleotides containing an α-l-RNA monomer and complementary RNA were good substrates for Escherichia coli RNase H. RNA-selective hybridization was also achieved by the incorporation of 1-(4-C-hydroxymethyl-β-d-lyxofuranosyl)thymine monomers into a DNA strand, whereas stable duplexes were formed with both complementary DNA and RNA when these monomers were incorporated into an RNA strand.     

Hydrophobic, Non-Hydrogen-Bonding Bases and Base Pairs in DNA

We report the properties of hydrophobic isosteres of pyrimidines and purines in synthetic DNA duplexes. Phenyl nucleosides 1 and 2 are nonpolar isosteres of the natural thymidine nucleoside, and indole nucleoside 3 is an analog of the complementary purine 2-aminodeoxyadenosine. The nucleosides were incorporated into synthetic oligodeoxynucleotides and were paired against each other and against the natural bases. Thermal denaturation experiments were used to measure the stabilities of the duplexes at neutral pH. It is found that the hydrophobic base analogs are nonselective in pairing with the four natural bases but selective for pairing with each other rather than with the natural bases. For example, compound 2 selectively pairs with itself rather than with A, T, G, or C; the magnitude of this selectivity is found to be 6.5-9.3 °C in Tm or 1.5-1.8 kcal/mol in free energy (25 °C). All possible hydrophobic pairing combinations of 1, 2, and 3 were examined. Results show that the pairing affinity depends on the nature of the pairs and on position in the duplex. The highest affinity pairs are found to be the 1-1 and 2-2 self-pairs and the 1-2 heteropair. The best stabilization occurs when the pairs are placed at the ends of duplexes rather than internally; the internal pairs may be destabilized by imperfect steric mimicry which leads to non-ideal duplex structure. In some cases the hydrophobic pairs are significantly stabilizing to the DNA duplex; for example, when situated at the end of a duplex, the 1-1 pair is more stabilizing than a T-A pair. When situated internally, the affinity of the 1-1 pair is the same as, or slightly better than, the analogous T-T mismatch pair, which is known to have two hydrogen bonds. The studies raise the possibility that hydrogen bonds may not always be required for the formation of stable duplex DNA-like structure. In addition, the results point out the importance of solvation and desolvation in natural base pairing, and lend new support to the idea that hydrogen bonds in DNA may be more important for specificity of pairing than for affinity. Finally, the study raises the possibility of using these or related base pairs to expand the genetic code beyond the natural A-T and G-C pairs.     

Synthesis and stability of oligodeoxynucleotides containing C8-labeled 2'-deoxyadenosine: novel redox nucleobase probes for DNA-mediated charge-transfer studies

[reaction: see text] An efficient and convenient synthetic strategy to redox-labeled C8-derivatives of 2'-deoxyadenosine is described. The Pd(0) cross-coupling chemistry is amenable to both oxidative and reductive redox probes. The corresponding phosphoramidites of phenothiazine and anthraquinone nucleosides are amenable to automated DNA synthesis. The resulting labeled oligodeoxynucleotide strands form stable B-form duplexes with melting temperatures and CD spectra similar to those of the unlabeled analogues.     

Perylene-3,4:9,10-tetracarboxylic acid bisimide dye as an artificial DNA base surrogate

A phosphoramidite of the perylene bisimide dye was synthesized as a DNA building block that allows incorporation of this chromophore as an artificial nucleoside surrogate either at the 5'-terminus or at internal positions of duplex DNA. The internally incorporated perylene bisimide chromophore shows strong interactions with the DNA base stack; the 5'-terminally attached perylene bisimide is able to induce dimerization of two whole DNA duplexes.     

功能化纳米金放大的DNA电化学传感器研究

研究了DNA夹心杂交和直接杂交体系,将功能化纳米金引入到标记有生物素的杂交双链上,制成具有电化学活性和纳米金放大作用的DNA电化学传感器,采用循环伏安法测试.在夹心杂交体系中,靶点DNA浓度与阳极峰电流关系曲线的相对标准偏差为3.0%～13.0%,在浓度为6.9×10^-3～0.14nmol/L范围内得到良好的线性关系,检测限达到2.0×10^-3nmol/L,实现了对单碱基突变的高灵敏检测和序列识别.直接杂交检测限为2.5×10^-4mol/L,分别在2.5×10^-4～5.0×10^-3nmol/L和5.0×10^-3～10nmol/L范围内得到峰电量与浓度的良好线性关系.并比较这两种体系.     

Measurement of nucleobase pKa values in model mononucleotides shows RNA-RNA duplexes to be more stable than DNA-DNA duplexes

To understand why the RNA-RNA duplexes in general has a higher thermodynamic stability over the corresponding DNA-DNA duplexes, we have measured the pK(a) values of both nucleoside 3',5'-bis-ethyl phosphates [Etp(d/rN)pEt] and nucleoside 3'-ethyl phosphates [(d/rN)pEt] (N = A, G, C, or T/U), modeling as donors and acceptors of base pairs in duplexes. While the 3',5'-bis-phosphates, Etp(d/rN)pEt, mimic the internucleotidic monomeric units of DNA and RNA, in which the stacking contribution is completely absent, the 3'-ethyl phosphates, (d/rN)pEt, mimic the nucleotide at the 5'-end. The pK(a) values of the nucleobase in each of these model nucleoside phosphates have been determined with low pK(a) error (sigma = +/-0.01 to 0.02) by (1)H NMR (at 500 MHz) with 20-33 different pH measurements for each compound. This study has led us to show the following: (1) All monomeric DNA nucleobases are more basic than the corresponding RNA nucleobases in their respective Etp(d/rN)pEt and (d/rN)pEt. (2) The pK(a) values of the monomeric nucleotide blocks as well as Delta pK(a) values between the donor and acceptor can be used to understand the relative base-pairing strength in the oligomeric duplexes in the RNA and DNA series. (3) The Delta G*(pKa) of the donor and acceptor of the base pair in duplexes enables a qualitative dissection of the relative strength of the base-pairing and stacking in the RNA-RNA over the DNA-DNA duplexes. (4) It is also found that the relative contribution of base-pairing strength and nucleobase stacking in RNA-RNA over DNA-DNA is mutually compensating as the % A-T/U content increases or decreases. This interdependency of stacking and hydrogen bonding can be potentially important in the molecular design of the base-pair mimics to expand the alphabet of the genetic code.     

Ligand-based backbone modifications for metal-chelating nucleic acids

Ligands were incorporated into the backbone of DNA for nucleoside replacements, and the binding of metal ions, such as Cu2+, Pt2+ and Pd4+, was shown to influence stability of the resulting duplexes.     

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.     

Synthesis and characterization of DNA duplexes containing an N(4)C-ethyl-N(4)C interstrand cross-link

Short DNA duplexes containing an N(4)C-ethyl-N(4)C interstrand cross-link, C-C, were synthesized on controlled pore glass supports. Duplexes having two, three, or four A/T base pairs on either side of the C-C cross-link and terminating with a C(4) overhang at their 5'-ends were prepared. The cross-link was introduced using a convertible nucleoside approach. Thus, an oligonucleotide terminating at its 5'-end with O(4)-triazoyl-2'-deoxyuridine was first prepared on the support. The triazole group of support-bound oligomer was displaced by the aminoethyl group of 5'-dimethoxytrityl-3'-O-tert-butyldimethylsilyl-N(4)-(2-aminoethyl)deoxycytidine to give the cross-link. The dimethoxytrityl group was removed, and the upper and lower strands of the duplex were extended from two 5'-hydroxyl groups of the cross-link using protected nucleoside 3'-phosphoramidites. The tert-butyldimethylsilyl group of the resulting partial duplex was then removed, and the chain was extended in the 3'-direction from the resulting 3'-hydroxyl of the cross-link using protected nucleoside 5'-phosphoramidites. The cross-linked duplexes were purified by HPLC and characterized by enzymatic digestion and MALDI-TOF mass spectrometry. Duplexes with three or four A/T base pairs on either side of the C-C cross-link gave sigmoidal shaped A(260) profiles when heated, a behavior consistent with cooperative denaturation of the A/T base pairs. Each cross-linked duplex could be ligated to an acceptor duplex using T4 DNA ligase, a result that suggests that the C-C cross-link does not interfere with the ligation reaction, even when it is located only two base pairs from the site of ligation. The ability to synthesize duplexes with a defined interstrand cross-link and to incorporate these duplexes into longer pieces of DNA should enable preparation of substrates that can be used for a variety of biophysical and biochemical experiments, including studies of DNA repair.     

New base pairing motifs. The synthesis and thermal stability of oligodeoxynucleotides containing imidazopyridopyrimidine nucleosides with the ability to form four hydrogen bonds

The synthesis and thermal stability of oligodeoxynucleotides (ODNs) containing imidazo[5',4':4,5]pyrido[2,3-d]pyrimidine nucleosides 1-4 (N(N), O(O), N(O), and O(N), respectively) with the aim of developing two sets of new base pairing motifs consisting of four hydrogen bonds (H-bonds) is described. The proposed four tricyclic nucleosides 1-4 were synthesized through the Stille coupling reaction of a 5-iodoimidazole nucleoside with an appropriate 5-stannylpyrimidine derivative, followed by an intramolecular cyclization. These nucleosides were incorporated into ODNs to investigate the H-bonding ability. When one molecule of the tricyclic nucleosides was incorporated into the center of each ODN (ODN I and II, each 17mer), no apparent specificity of base pairing was observed, and all duplexes were less stable than the duplexes containing natural G:C and A:T pairs. On the other hand, when three molecules of the tricyclic nucleosides were consecutively incorporated into the center of each ODN (ODN III and IV, each 17mer), thermal and thermodynamic stabilization of the duplexes due to the specific base pairings was observed. The melting temperature (T(m)) of the duplex containing the N(O):O(N) pairs showed the highest T(m) of 84.0 degrees C, which was 18.2 and 23.5 degrees C higher than that of the duplexes containing G:C and A:T pairs, respectively. This result implies that N(O)and O(N) form base pairs with four H-bonds when they are incorporated into ODNs. The duplex containing N(O):O(N) pairs was markedly stabilized by the assistance of the stacking ability of the imidazopyridopyrimidine bases. Thus, we developed a thermally stable new base pairing motif, which should be useful for the stabilization and regulation of a variety of DNA structures.