Thermal Stability Of Duplexes Research Articles

Microfluidic Thermodynamics of the Shift in Thermal Stability of DNA Duplex in a Microchannel Laminar Flow

This paper reports the shift in thermal stability of DNA duplex and its thermodynamics spectroscopically, caused by stretching and orientation of DNA strands in a microchannel laminar flow. For direct spectroscopic measurement of the microchannel, we prepared an in-house temperature-controllable microchannel-type flow cell. The melting curves of DNA oligomers in a microchannel laminar flow were measured. For DNA oligomers with more than 10 base pairs, the melting curve shifted to the high-temperature side with higher flow speed. However, for 8-base-pair DNA oligomers, a change in the melting profile was not observed in batchwise and microchannel flows. We undertook microfluidic thermodynamic analysis to elucidate details of the shift in thermal stability of the DNA duplex in a microchannel laminar flow. Enthalpy-entropy compensation is applicable to the microfluidic thermal stability shift. We studied the relationships between the enthalpy-entropy compensation and DNA strand length or flow speed. Results showed that the enthalpy-entropy compensation was influenced by both DNA strand length and flow speed, and the penalties of enthalpy were 2-12% greater than the benefits of entropy.

A fluorescence-quencher pair for DNA hybridization studies based on hydrophobic base surrogates

The synthesis of the novel, fluorescent 3-aminobiphenyl-C-nucleoside M as well as the corresponding building block for oligodeoxynucleotide synthesis is described. M was incorporated into oligodeoxynucleotides via standard phosphoramidite chemistry and the thermal stabilities of duplexes with one and three consecutive M– M, M– P, and M– O pairs, where P denotes an unmodified biphenyl C-nucleoside and O a 3,5-dinitrobiphenyl-C-nucleoside, were determined. It was found that duplexes containing three consecutive M– O pairs were the most stable in the series, notably more stable than a duplex with one additional natural G–C pair instead of the modified residues. Furthermore it was found that the fluorescence of M is efficiently quenched in a duplex when placed opposite to the dinitrophenyl unit O. Thus M and O constitute a novel fluorophore/quencher pair that is orthogonal to natural base pairs in its recognition properties enabling its use as highly specific tags with reporting properties.

DNA and LNA oligonucleotides containing N2′-functionalised derivatives of 2′-amino-2′-deoxyuridine

Synthesis of various N-acylated derivatives of 2′-amino-2′-deoxyuridine is described together with their incorporation into DNA and LNA oligonucleotides using the phosphoramidite approach on an automated DNA synthesizer. The thermal stabilities of duplexes formed by these 2′-amino-DNA-modified DNA or LNA/DNA chimeric strands and complementary DNA or RNA strands have been studied. Introduction of LNA monomers around the functionalised 2′-amino-DNA modifications results in reversal of the affinity-decreasing effect of the latter. This represents a novel general approach for design and synthesis of high-affinity functionalised oligonucleotides for biotechnological or medicinal applications.

Polyaza Crown Ethers as Non‐Nucleosidic Building Blocks in DNA Conjugates: Synthesis and Remarkable Stabilization of dsDNA

The synthesis of amphiphilic polyaza crown ether monomers X (benzyl-substituted), Y (palmityl-substituted) and Z (cholesteryl-substituted) and their incorporation into oligonucleotides are described. Their effects on thermal duplex stability were investigated by UV melting curve analysis in different alkaline metal buffer solutions. Thermal-denaturation experiments showed remarkable stabilization of dsDNA by polyaza crown ether monomers when incorporated in opposite positions. The series of polyaza crown ether monomers (X, Y and Z) with different overall lipophilicities showed a trend of increased stability of the corresponding dsDNA with increasing lipophilicity of the polyaza crown ether monomer. Multiple incorporations of benzyl-substituted polyaza crown ether monomer X as dangling ends on both sides of dsDNA resulted in strongly increased stability of the corresponding duplex.

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.

Synthesis and Properties of Oligodeoxyribonucleotides Containing 2′‐O‐(2,3‐Dihydroxypropyl)‐ and 2′‐O‐(2‐Oxoethyl)arabinouridine Residues.

2’-O-(2,3-Dihydroxypropyl)arabinouridine-containing oligodeoxyribonucleotides were synthesized starting from a new modified nucleoside, viz., 2’-O-(2,3-dihydroxypropyl)arabinouridine, and the corresponding 3’-phosphoramidite. Oxidation of these oligodeoxyribonucleotides with sodium periodate afforded oligonucleotides containing 2’-O-(2-oxoethyl)arabinouridine residues. Subsequent modification of the aldehyde-containing oligonucleotides involved the reactions with 9-hydrazinoacridine and N-aminooxyacetyl peptide and reductive amination by 4-(1-pyrenyl)butyrohydrazide and biotin hydrazide. Thermal stabilities of duplexes of modified oligodeoxyribonucleotides with complementary oligodeoxyribonucleotides are slightly lower than those of natural duplexes. Duplexes with complementary oligoribonucleotides are substantially destabilized.

Investigating HIV-1 Polypurine Tract Geometry via Targeted Insertion of Abasic Lesions in the (–)-DNA Template and (+)-RNA Primer

A variety of biochemical and structural studies indicate that two regions of the human immunodeficiency virus type 1 (HIV-1) polypurine tract (PPT)-containing RNA/DNA hybrid deviate from standard Watson-Crick geometry. However, it is unclear whether and how these regions cooperate to ensure PPT primer selection by reverse transcriptase-associated ribonuclease H and subsequent removal from nascent (+)-DNA. To address these issues, we synthesized oligonucleotides containing abasic lesions in either the PPT (+)-RNA primer or (-)-DNA template to locally remove nucleobases, although retaining the sugar-phosphate backbone. KMnO(4) footprinting indicates such lesions locally alter duplex structure, whereas thermal melting studies show significantly reduced stability when lesions are positioned around the scissile bond. Substituting the (-)-DNA template between positions -15 and -13 altered cleavage specificity, whereas equivalent substitutions of the (+)-RNA had almost no effect. The unpaired base of the DNA template observed crystallographically (-11C) could also be removed without significant loss of cleavage specificity. With respect to the scissile -1/+1 phosphodiester bond, template nucleobases could be removed without loss of cleavage specificity, whereas equivalent lesions in the RNA primer were inhibitory. Our data suggest an interaction between the p66 thumb subdomain of HIV-1 reverse transcriptase, and the DNA template in the "unzipped" portion of the RNA/DNA hybrid could aid in positioning the ribonuclease H catalytic center at the PPT/U3 junction and also provides insights into nucleic acid geometry around the scissile bond required for hydrolysis.

The effect of the 2-amino group of 7,8-dihydro-8-oxo-2'-deoxyguanosine on translesion synthesis and duplex stability

Replication of DNA containing 7,8-dihydro-8-oxo-2′-deoxyguanosine (OxodG) gives rise to G → T transversions. The syn-isomer of the lesion directs misincorporation of 2′-deoxyadenosine (dA) opposite it. We investigated the role of the 2-amino substituent on duplex thermal stability and in replication using 7,8-dihydro-8-oxo-2′-deoxyinosine (OxodI). Oligonucleotides containing OxodI at defined sites were chemically synthesized via solid phase synthesis. Translesion incorporation opposite OxodI was compared with 7,8-dihydro-8-oxo-2′-deoxyguanosine (OxodG), 2′-deoxyinosine (dI) and 2′-deoxyguanosine (dG) in otherwise identical templates. The Klenow exo− fragment of Escherichia coli DNA polymerase I incorporated 2′-deoxyadenosine (dA) six times more frequently than 2′-deoxycytidine (dC) opposite OxodI. Preferential translesion incorporation of dA was unique to OxodI. UV-melting experiments revealed that DNA containing OxodI opposite dA is more stable than when the modified nucleotide is opposed by dC. These data suggest that while duplex DNA accommodates the 2-amino group in syn-OxodG, this substituent is thermally destabilizing and does not provide a kinetic inducement for replication by Klenow exo−.

Bipyridyl‐ and Biphenyl‐DNA: A Recognition Motif Based on Interstrand Aromatic Stacking

The synthesis and incorporation into oligonucleotides of C-nucleosides containing the two aromatic, non-hydrogen-bonding nucleobase substitutes biphenyl (I) and bipyridyl (Y) are described. Their homo- and hetero-recognition properties in different sequential arrangements were then investigated via UV-melting curve analysis, gel mobility assays, CD- and NMR spectroscopy. An NMR analysis of a dodecamer duplex containing one biphenyl pair in the center, as well as CD data on duplexes with multiple insertions provide further evidence for the zipper-like interstrand stacking motif that we proposed earlier based on molecular modeling. UV-thermal melting experiments with duplexes containing one to up to seven I- or Y base pairs revealed a constant increase in T(m) in the case of I and a constant decrease for Y. Mixed I/Y base pairs lead to stabilities in between the homoseries. Insertion of alternating I/abasic site- or Y/abasic site pairs strongly decreases the thermal stability of duplexes. Asymmetric distribution of I- or Y residues on either strand of the duplex were also investigated in this context. Duplexes with three natural base pairs at both ends and 50 % of I pairs in the center are still readily formed, while duplexes with blunt ended I pairs tend to aggregate unspecifically. Duplexes with one natural overhang at the end of a I-I base pair tract can both aggregate or form ordered duplexes, depending on the nature of the natural bases in the overhang.

Synthesis and properties of oligodeoxyribonucleotides containing 2’-O-(2,3-dihydroxypropyl)- and 2’-O-(2-oxoethyl)arabinouridine residues

2’-O-(2,3-Dihydroxypropyl)arabinouridine-containing oligodeoxyribonucleotides were synthesized starting from a new modified nucleoside, viz., 2’-O-(2,3-dihydroxypropyl)arabinouridine, and the corresponding 3’-phosphoramidite. Oxidation of these oligodeoxyribonucleotides with sodium periodate afforded oligonucleotides containing 2’-O-(2-oxoethyl)arabinouridine residues. Subsequent modification of the aldehyde-containing oligonucleotides involved the reactions with 9-hydrazinoacridine and N-aminooxyacetyl peptide and reductive amination by 4-(1-pyrenyl)butyrohydrazide and biotin hydrazide. Thermal stabilities of duplexes of modified oligodeoxyribonucleotides with complementary oligodeoxyribonucleotides are slightly lower than those of natural duplexes. Duplexes with complementary oligoribonucleotides are substantially destabilized.

Oligonucleotide–Minor Groove Binder Conjugates and Their Complexes with Complementary DNA: Effect of Conjugate Structural Factors on the Thermal Stability of Duplexes

Synthetic polycarboxamide minor groove binders (MGB) consisting of N‐methylpyrrole (Py), N‐methylimidazole (Im), N‐methyl‐3‐hydroxypyrrole (Hp) and β‐alanine (β) show strong and sequence‐specific interaction with the DNA minor groove in side‐by‐side antiparallel or parallel orientation. Two MGB moieties covalently linked to the same terminal phosphate of one DNA strand stabilize DNA duplexes formed by this strand with a complementary one in a sequence‐specific manner, similarly to the corresponding mono‐conjugated hairpin structures. The series of conjugates with the general formula Oligo‐(L‐MGB‐R)m was synthesized, where m = 1 or 2, L = linker, R = terminal charged or neutral group, MGB = –(Py)n–, –(Im)n– or –[(Py/Im)n–(CH2)3CONH–(Py/Im)n–] and 1 < n < 5. Using thermal denaturation, we studied effects of structural factors such as m and n, linker L length, nature and orientation of the MGB monomers, the group R and the backbone (DNA or RNA), etc. on the stability of the duplexes. Structural factors are more important for linear and hairpin monophosphoroamidates than for parallel bis‐phosphoroamidates. No more than two oligocarboxamide strands can be inserted into the duplex minor groove. Attachment of the second sequence‐specific parallel ligand [–L(Py)4R] to monophosphoroamidate conjugate CGTTTATT–L(Py)4R leads to the increase of the duplex Tm, whereas attachment of [–L(Im)4R] leads to its decrease. The mode of interaction between oligonucleotide duplex and attached ligands could be different (stacking with the terminal A:T pair of the duplex or its insertion into the minor groove) depending on the length and structure of the MGB.

LNA-modified oligonucleotides are highly efficient as FISH probes

Fluorescence in situ hybridization (FISH) is a highly useful technique with a wide range of applications including the delineation of complex karyotypes, prenatal diagnosis of aneuploidies, screening for diagnostic or prognostic markers in cancer cells, gene mapping and gene expression studies. However, it is still a fairly time-consuming method with limitations in both sensitivity and resolution. Locked Nucleic Acids (LNAs) constitute a novel class of RNA analogs that have an exceptionally high affinity towards complementary DNA and RNA. Substitution of DNA oligonucleotide probes with LNA has shown to significantly increase their thermal duplex stability as well as to improve the discrimination between perfectly matched and mismatched target nucleic acids. To exploit the improved hybridization properties of LNA oligonucleotides in FISH, we have designed several LNA substituted oligonucleotide probes specific to different human-specific repetitive elements, such as the classical satellite-2, telomere and alpha-satellite repeats. In the present study we show that LNA modified oligonucleotides are excellent probes in FISH, combining high binding affinity with short hybridization time.

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 properties of RNA analogues having amides as interuridine linkages at selected positions.

Oligoribonucleotide analogues having amide internucleoside linkages (AM1: 3'-CH(2)CONH-5' and AM2: 3'-CH(2)NHCO-5') at selected positions have been synthesized and the thermal stability of duplexes formed by these analogues with complementary RNA fragments has been evaluated by UV melting experiments. Two series of oligomers with either 2'-OH or 2'-OMe vicinal to the amide linkages were studied. Monomeric synthons (3' and 5'-C amines and carboxylic acids) were synthesized as follows: For synthesis of the AM1 analogue, the known sequence of radical allylation followed by the cleavage of the double bond was adopted. For synthesis of the AM2 analogue, novel routes via addition of nitromethane followed by conversion of the nitro function to either amino or carboxyl groups were developed. Coupling of monomeric amines and carboxylic acids followed by protecting group manipulation and phosphonylation gave dimeric 3'-hydrogenphosphonate building blocks for oligonucleotide synthesis. Monomeric model compounds having 3'-amide and 2'-OH or 2'-OMe groups were also prepared and their conformational equilibrium was determined by (1)H NMR. The AM1 and AM2 models showed equal preferences for the North conformers (at 40 degrees C, 88-89% with 2'-OH, and 92-93% with 2'-OMe). At physiological salt concentration (0.1 M NaCl) the duplexes between AM1 modified oligonucleotides and RNA had stability similar to unmodified RNA-RNA duplexes (Delta t(m)= -0.2 to +0.7 degrees C per modification). However, the AM2 modification resulted in substantial stabilization of duplexes: Delta t(m)= +1 to +2.4 degrees C per modification compared to all RNA. A 2'-O-methyl vicinal to the AM2 linkage further increased the duplex stability. Our results suggest that RNA analogues having amide internucleoside bonds are very promising candidates for medicinal applications.

Fluorescence properties, thermal duplex stability, and kinetics of formation of cyanin platinum DNAs

Fluorescent and haptenized, monofunctionally binding platinum compounds are increasingly used for chemically labeling nucleic acids for hybridization detection purposes. Here we analyze in detail the effect of labeling density of the cyanin-3 and -5 platinum DNA adducts on fluorescence and thermal stability. We also analyzed the kinetics of the reaction of the cyanin platinum compounds with DNA. The data provided are important for the design of optimal platinum DNA labeling and hybridization conditions for fluorescence hybridization applications.

Synthesis and Incorporation into PNA of Fluorinated Olefinic PNA (F-OPA) Monomers

[structure: see text] A fluorinated OPA monomer containing the base thymine ((Z)-t-F-OPA) was synthesized in 12 steps, featuring a highly selective allylic over homoallylic Mitsunobu substitution for the introduction of the nucleobase. F-OPA modified PNA decamers were prepared by the MMTr/acyl protection strategy. The thermal stability of duplexes of PNA decamers containing (Z)-t-F-OPA units with antiparallel complementary DNA was measured. We found a strong dependence of stability from the sequential position of the (Z)-t-F-OPA units, ranging from DeltaT(m) of +2.4 to -8.1 degrees C/modification relative to unmodified PNA.

Design and test of noncrosshybridizing oligonucleotide building blocks for DNA computers and nanostructures

DNA oligonucleotides that anneal to form duplexes in specific, planned configurations are a basic construction material for DNA-based computers and nanotechnology. Unplanned duplex configurations introduce errors in computations and defects in structures, and thus, the sequences must be designed to minimize these effects. A software design tool has been developed that uses thermodynamic models of DNA duplex thermal stability and algorithms from graph theory to select good sets of oligonucleotides. An example set was tested in the laboratory, and the designed sequences formed no unplanned duplexes and had no detectable secondary structure.

Synthesis of oligonucleotides and thermal stability of duplexes containing the beta-C-nucleoside analogue of Fapy*dG.

The formamidopyrimidine lesions (Fapy.dA, Fapy.dG) are formed in significant amounts when DNA is exposed to oxidative stress. These lesions are unusual in that they readily epimerize in solution. The distribution of configurational isomers in DNA is unknown. Nonepimerizable, nonhydrolyzable analogues are useful probes for investigating the configuration of Fapy lesions in DNA and as potential enzyme inhibitors. The beta-C-nucleoside of Fapy.dG has been prepared and introduced sight-specifically into oligonucleotides via its respective beta-cyanoethyl phosphoramidite. The phosphoramidite was prepared via a Wittig reaction between a protected form of deoxyribose and a suitably functionalized pyrimidine. The pyrimidine contained methyl and 2-propyl groups at the O4 and O2 positions, respectively, to differentiate between them following C-nucleoside formation. The formamide was derived from a nitro group at C5. The phosphoramidite coupled in 80% yield via a single 15-min coupling using tetrazole as activator. Oligonucleotides as long as 36 nucleotides were prepared and characterized by ESI-MS.

The influence of various modified nucleotides placed as 3′-dangling end on thermal stability of RNA duplexes

The ribonucleic acids (RNA) form highly folded structures, which behind the helical fragments contain several secondary and tertiary structural motives. All of them have an influence on thermodynamic stability of the RNA. The 5′- and 3′-dangling ends are one of those structural motives, which effect stability of the adjacent helixes. In this paper, we described the influence of 14 different modified nucleotides, placed as 3′-dangling ends, on thermal stability of the RNA duplexes. Collected data demonstrate that: (i) 5-substituents of the uridine have an impact on the 3′-dangling end effect and the largest changes were observed for 5-chloro, bromo and methyl substituents; (ii) position of the methyl group within the uracil residue affect the thermal stability of the duplex; (iii) increasing a size of the heterocycle base placed as the 3′-terminal unpaired nucleotide enhances stabilization of duplexes.

Synthesis and characterization of site-specific and stereoisomeric fjord dibenzo[a,l]pyrene diol epoxide-N(6)-adenine adducts: unusual thermal stabilization of modified DNA duplexes.

The fjord polycyclic aromatic hydrocarbon compound dibenzo[a,l]pyrene (DB[a,l]P) is significantly more tumorigenic than the bay region benzo[a]pyrene in animal model systems. The molecular origins of the unusually strong genotoxic properties of DB[a,l]P and its fjord region diol epoxide metabolites are of great interest and are believed to be related to the structural characteristics of the DNA adducts formed. Site-specifically modified oligonucleotides were prepared by reacting the single adenine residue in 5'-d(CTCTCACTTCC) (I) with the racemic fjord diol epoxide r11,t12-dihydrodiol-t13,14-epoxide-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (anti-DB[a,l]PDE) in aqueous solutions. Four different oligonucleotides I with the single adenosine residues involving a covalent bond between the C14 position of DB[a,l]PDE and N(6)-dA are identified and purified. The CD spectra of the mononucleotide adducts are similar to those of Li et al. [Li et al. (1999) Chem. Res. Toxicol. 12, 758] who characterized DB[a,l]PDE-N(6)-dA adducts by a combination of CD and NMR methods. The stereochemical properties of each of the four DB[a,l]PDE-modified oligonucleotides were assigned on the basis of a combination of empirical CD rules and other approaches and differ from those of Li et al. The thermal melting points, T(m), of the unmodified duplex of I with its complementary strand (IC), T(m) = 43.8 +/- 0.5 degrees C, were compared with the same duplexes containing stereoisomeric anti-DB[a,l]PDE-N(6)-dA lesions. The T(m) of duplexes I.IC containing lesions with R absolute configurations at C14 of the DB[a,l]PDE residues are greater by 6-8 degrees C, while those with S configuration are lower by 6-10 degrees C. Similar effects are observed with adducts in the same sequence context derived from the fjord PAH anti-diol epoxides of benzo[g]chrysene, while duplexes containing lesions derived from benzo[c]phenanthrene diol epoxides with 1R and 1S configurations exhibit unchanged T(m) values. In contrast, the T(m) values of duplexes with lesions derived from the bay region benzo[a]pyrene diol epoxides (B[a]PDE) in the same sequence are lower by 12 degrees (10R adducts) and by 19 degrees (10S adducts). The greater thermal stabilities of duplexes with fjord PAH-N(6)-dA lesions relative to those with bay region B[a]PDE-N(6)-dA adducts, are correlated with lower susceptibilities of excision by human nucleotide excision repair enzymes [Buterin et al. (2000) Cancer Res. 60, 1849]. The implications of these relationships are discussed in terms of present knowledge of the conformations of fjord and bay region PAH diol epoxide-N(6)-dA lesions in double stranded DNA.