Endcaps for stabilizing short DNA duplexes

The syntheses of endcaps for covalently linking the 3' and 5' hydroxyl groups of blunt end double-stranded DNA are described. Endcap diols were converted into DMTr protected phosphoramidites and incorporated between nucleotides 4 and 5 of a self-complementary octamer. The stabilizing effect of the endcaps on duplex DNA was determined by Tm experiments on the self-complementary octamer.     

Effect of DNA flanking sequence on charge transport in short DNA duplexes

Several factors can influence charge transport (CT)-mediated DNA, such as sequence, distance, base stacking, base pair mismatch, conformation, tether length, etc. However, the DNA context effect or how flanking sequences influence redox active drugs in the DNA CT reaction and later in DNA enzymatic repair and synthesis is still not well understood. The set of seven DNA molecules in this study have been characterized well for the study of flanking sequence effects. These DNA duplexes are formed from self-complementary strands and contain the common central four-base sequence 5'-A-G-C-T-3', flanked on both sides by either (AT)(n) or (AA)(n) (n = 2, 3, or 4) or AA(AT)(2). UV-vis, fluorescence, UV melting, circular dichroism, and cyclic voltammetry experiments were used to study the flanking sequence effect on CT-mediated DNA by using daunomycin or adriamycin cross-linked with these seven DNA molecules. Our results showed that charge transport was related to the flanking sequence, DNA melting free energy, and ionic strength. For (AA)(n) or (AT)(n) species of the same length, (AA)(n) series were more stable and more efficient CT was observed through the (AA)(n) series. The same trend was observed for (AA)(n)() and (AT)(n) series at different ionic strengths, further supporting the idea that flanking sequence can result in different base stacking and modulate charge transport through these seven DNA molecules.     

Electron transfer in DNA duplexes containing 2-methyl-1,4-naphthoquinone

2-Methyl-1,4-naphthoquinone (menadione, MQ) was linked to synthetic oligonucleotides and exposed to near-UV light to generate base radical cations in DNA. This model system of electron transfer induced alkali-labile breaks at GG doublets, similar to anthraquinone and metallointercalators systems. In sharp contrast to other systems, the photolysis of MQ–DNA duplexes gave interstrand cross-links and alkali-labile breaks at bases on the complementary strand opposite the MQ moiety. For sequences with an internal MQ, the formation of cross-links with A and C opposite the MQ moiety was 2- to 3-fold greater than that with G and T. The yield of cross-links was more than 10-fold greater than that of breaks opposite MQ, which in turn was more than 2-fold greater than breaks at GG doublets. The yield of damage at GG doublets greatly increased for a sequence with a terminal MQ. The distribution of base damage was measured by enzymatic digestion and HPLC analysis (dAdo > dThd > dGuo > dCyd). The formation of novel products in MQ–DNA duplexes was attributed to the ability of excited MQ to generate the radical cations of all four DNA bases; thus, this photochemical reaction provides an ideal model system to study the effects of ionizing radiation and one-electron oxidants.     

Thermal stabilisation of the short DNA duplexes by acridine-4-carboxamide derivatives

The short oligodeoxynucleotide (ODN) probes are suitable for good discrimination of point mutations. However, the probes suffer from low melting temperatures. In this work, the strategy of using acridine-4-carboxamide intercalators to improve thermal stabilisation is investigated. The study of large series of acridines revealed that optimal stabilisation is achieved upon decoration of acridine by secondary carboxamide carrying sterically not demanding basic function bound through a two-carbon linker. Two highly active intercalators were attached to short probes (13 or 18 bases; designed as a part of HFE gene) by click chemistry into positions 7 and/or 13 and proved to increase the melting temperate (T_m) of the duplex by almost 8°C for the best combination. The acridines interact with both single- and double-stranded DNAs with substantially preferred interaction for the latter. The study of interaction suggested higher affinity of the acridines toward the GC- than AT-rich sequences. Good discrimination of two point mutations was shown in practical application with HFE gene (wild type, H63D C > G and S65C A > C mutations). Acridine itself can also serve as a fluorophore and also allows discrimination of the fully matched sequences from those with point mutations in probes labelled only with acridine.     

T4 polynucleotide ligase catalyzed joining of short synthetic DNA duplexes at base-paired ends

The self-complementary octanucleotide dT-A-G-T-A-C-T-A has been synthesized and its sequence confirmed by two-dimensional fingerprinting. Under conditions used for the T4 polynucleotide ligase reaction, this oligonucleotide forms a dimeric duplex which shows a Tm of 18 degrees C. The optimal rate of joining of the 32P-labeled duplex occurs between 12 and 15 degrees C. The rate is highly concentration dependent, as expected for a bimolecular process. Polyacrylamide gel electrophoretic analysis of this reaction shows the presence of products up to 120 nucleotides in length. In a denaturing gel, each product appears as a double band due to the presence of its 5'-adenylylated activated intermediate. Substrates larger than eight base pairs are utilized more rapidly than the eight base pair duplex, indicating that the T4 ligase has a higher affinity for longer substrates. The low level of nicked intermediates suggests that the joining of both strands requires two steps, the rates of which must be similar.     

DNA secondary structure of the released strand stimulates WRN helicase action on forked duplexes without coordinate action of WRN exonuclease

Werner syndrome (WS) is an autosomal recessive premature aging disorder characterized by aging-related phenotypes and genomic instability. WS is caused by mutations in a gene encoding a nuclear protein, Werner syndrome protein (WRN), a member of the RecQ helicase family, that interestingly possesses both helicase and exonuclease activities. Previous studies have shown that the two activities act in concert on a single substrate. We investigated the effect of a DNA secondary structure on the two WRN activities and found that a DNA secondary structure of the displaced strand during unwinding stimulates WRN helicase without coordinate action of WRN exonuclease. These results imply that WRN helicase and exonuclease activities can act independently, and we propose that the uncoordinated action may be relevant to the in vivo activity of WRN.     

Conformation and dynamics of short DNA duplexes: (dC-dG)3 and (dC-dG)4

Natural abundance 13C NMR spectra of duplexed (dC-dG)3 and (dC-dG)4 exhibit resolved resonances for most of the carbons at 0.1M NaCl in aqueous solution. Large transitions in chemical shift for many of the hexamer carbons (up to 1.8 ppm) are observed in variable temperature measurements. Determination of spin-lattice relaxation times and nuclear Overhauser enhancements in 0.1M NaCl indicate that the duplexes tumble almost isotropically, with overall correlation times near 5 nsec; the sugar carbons experience more rapid local motions than do the base carbons. The relaxation data are also consistent with the most rapid local motions occurring at the chain-terminal residues, especially in the Cyd(1) sugar. 4M NaCl causes changes in the 13C chemical shifts of most of the guanine base carbons, and rearrangements in the deoxyribose carbon shifts; this is consistent with changes predicted by a salt-induced B to Z transition, viz. conversion of the guanylates from the anti to syn range about the glycosyl bond, and from the S to N pseudorotational state of the deoxyribose ring.     

Mutational analysis reveals two independent molecular requirements during transfer RNA selection on the ribosome

Accurate discrimination between cognate and near-cognate aminoacyl-tRNAs during translation relies on the specific acceleration of forward rate constants for cognate tRNAs. Such specific rate enhancement correlates with conformational changes in the tRNA and small ribosomal subunit that depend on an RNA-specific type of interaction, the A-minor motif, between universally conserved 16S ribosomal RNA nucleotides and the cognate codon-anticodon helix. We show that perturbations of these two components of the A-minor motif, the conserved rRNA bases and the codon-anticodon helix, result in distinct outcomes. Although both cause decreases in the rates of tRNA selection that are rescued by aminoglycoside antibiotics, only disruption of the codon-anticodon helix is overcome by a miscoding tRNA variant. On this basis, we propose that two independent molecular requirements must be met to allow tRNAs to proceed through the selection pathway, providing a mechanism for exquisite control of fidelity during this step in gene expression.     

Comparison of thermodynamic stabilities between PNA (peptide nucleic acid)/DNA hybrid duplexes and DNA/DNA duplexes.

We have examined quantitatively stabilities of PNA/DNA hybrid duplexes with identical nearest-neighbor base pairs and compared stabilities between PNA/DNA and DNA/DNA. The average difference of stabilization energy of the short PNA/DNA was 0.9 kcal mol(-1), which suggests that the stability of the hybrids with identical nearest-neighbor base pairs can be predicted with the nearest-neighbor model as well as those of nucleic acid duplexes.     

Linking two DNA duplexes with a rigid linker for DNA nanotechnology

DNA has recently emerged as a promising material for the construction of nanosized architectures. Chemically modified DNA has been suggested to be an important component of such architectural building blocks. We have designed and synthesized a novel H-shaped DNA oligonucleotide dimer that is cross-linked with a structurally rigid linker composed of phenylene and ethynylene groups. A rotatable DNA unit was constructed through the self-assembly of this H-shaped DNA component and two complementary DNA oligonucleotides. In addition to the rotatable unit, a locked DNA unit containing two H-shaped DNA components was also constructed. As an example of an extended locked structure, a hexagonal DNA origami dimer and oligomer were constructed by using H-shaped DNA as linkers.     

Tracts of A.T base pairs retard the electrophoretic mobility of short DNA duplexes.

An investigation of the mobility of short duplexes of DNA -octamers and decamers-on polyacrylamide gels is presented, showing that molecules containing less than one helical turn exhibit sequence dependent mobilities. Analysis of chains with different sequences indicates that any arrangement of two or more adjacent A.T base pairs causes a duplex to move more slowly than does any combination of isolated A.T pairs This behavior appears to be an intrinsic property of these sequences, since the anomaly persists in the absence of magnesium or presence of spermine and is not due to strand dissociation. In two decamers we studied, the position of A.T tracts within a duplex can be shown to influence mobility: the sequence GA4T4C associated with bending or curvature of the helix axis when ligated into polymers migrates more slowly than the corresponding sequence GT4A4C, polymers of which migrate as linear B DNA.     

Thermodynamics of polymolecular duplexes between phosphate-methylated DNA and natural DNA

Phosphate-methylated (P.M.) DNA possesses a very high affinity for complementary natural DNA, as a result of the absence of interstrand electrostatic repulsions. In this study, a model system phosphate-methylated d[Cn] with natural d(Gk) (n less than k) is chosen for an investigation of the thermodynamic properties that determine duplex stability. The enthalpy change of a melting transition is shown to be considerably larger than is observed for corresponding natural DNA duplexes. It is found that delta Hn0 of GG/CC nearest neighbor pairwise interaction equals -15.6 kcal/mol, compared to -11.0 kcal/mol for the natural analog. The entropy change is strongly dependent on the length of the natural DNA strand and the number of phosphate-methylated DNA oligomers hybridized. The results are explained by means of a model in which a cooperative effect for subsequent hybridizations of phosphate-methylated DNA oligomers is assumed, thus giving additional stability.     

A note on crystal packing and global helix structure in short A-DNA duplexes

A simple relation exists between the packing density in crystals of short A-DNA duplexes and their global double-helical structure. The volume per nucleotide pair shows a linear inverse correlation with the mean displacement of base pairs from the best straight helix axis. The mean displacement is a measure of major groove depth and varies between -3.3 A and -4.9 A in A-form oligonucleotides analysed in the crystalline state. Since the mean displacement of base pairs from the helix axis determines other helical parameters such as base-pair longitudinal slide, its correlation with crystal packing is of considerable interest. The displacement-packing correlation is very clear for octamer duplexes which crystallize in three different lattices. Longer A-helical fragments sometimes deviate from the rule. It may be speculated whether A-form duplexes not completing a full helical turn are especially prone to distortions due to packing in crystals or arising from intermolecular contacts in solution.     

A neural network for predicting the stability of DNA/DNA duplexes

A back-propagation neural network method has been developed to predict the stability of DNA/DNA duplexes. Calculated Tm with the present parameters fits the experimental values within reasonable errors (AD = 1.86 K, SEP = 1.99151, R2 = 0.9894 for NN1; AD = 1.59667 K, SEP = 2.03824, R2 = 0.99371 for NN2) and it has the advantage that the determinations of thermodynamic parameters are not needed.     

Site-directed mutagenesis reveals the thermodynamic requirements for single-stranded DNA recognition by the telomere-binding protein Cdc13

The essential Saccharomyces cerevisiae protein Cdc13 binds the conserved single-stranded overhang at the end of telomeres and mediates access of protein complexes involved in both end-capping and telomerase activity. The single-stranded DNA-binding domain (ssDBD) of Cdc13 exhibits both high affinity (K(d) of 3 pM) and sequence specificity for the GT-rich sequences present at yeast telomeres. We have used the ssDBD of Cdc13 to understand the sequence-specific recognition of extended single-stranded DNA (ssDNA). The recent structure of the Cdc13 DNA-binding domain revealed that ssDNA is recognized by a large protein surface containing an oligonucleotide/oligosaccharide-binding fold (OB-fold) augmented by an extended 30-amino acid loop. Contacts to ssDNA occur via a contiguous surface of aromatic, hydrophobic, and basic residues. A complete alanine scan of the binding interface has been used to determine the contribution of each contacting side chain to binding affinity. Substitution of any aromatic or hydrophobic residue at the interface was deleterious to binding (20 to >700-fold decrease in binding affinity), while tolerance for replacement of basic residues was observed. The important aromatic and hydrophobic contacts are spread throughout the extended interface, indicating that the entire surface is both structurally and thermodynamically required for binding. While all of these contacts are important, several of the individual alanine substitutions that abolish binding cluster to one region of the protein surface. This region is vital for recognition of four bases at the 5' end of the DNA and constitutes a "hotspot" of binding affinity.     

Mutational analysis of F-pilin reveals domains for pilus assembly, phage infection and DNA transfer

The F-pilus has been implicated in recipient cell recognition during the establishment of a stable mating pair before conjugation as well as forming part of the conjugative pore for DNA transfer. The F-pilus is the site of attachment of the filamentous phages (M13, f1 and fd), which attach to the F-pilus tip, and the RNA phages, R17 and Qbeta, which attach to different sites exposed on the sides of the pilus. R17 has been shown to undergo eclipse, or capsid release, outside the cell on pili attached to cells. New and existing mutants of traA combined with natural variants of F-pilin were assayed for pilin stability and processing, pilus elongation, transfer, phage sensitivity and R17 eclipse. Phenotypes of these mutants indicated that the F-pilin subunit contains specific regions that can be associated with pilus assembly, phage sensitivity and DNA transport. Mutations involving lysines and phenylalanines within residues 45-60 suggest that these residues might participate in transmitting a signal down the length of the pilus that initiates DNA transfer or R17 eclipse.     

The requirements for conjugal DNA synthesis in the donor strain during flac transfer.

Although neither rifampicin nor spectinomycin had any effect on the frequency of Flac transfer by a sensitive donor, rifampicin but not spectinomycin prevented donor conjugal DNA synthesis as measured in matings between a dnaB donor and a tdk recipient. An untranslated RNA species is therefore probably required for this synthesis, although transfer took place even in its absence. Donor conjugal DNA synthesis was abolished in a dnaE donor, showing that DNA polymerase III is responsible for this process; again, plasmid DNA transfer was not affected.Flac mutants lacking the F pilus gave neither donor conjugal DNA synthesis nor plasmid DNA transfer, probably because they could not receive a “mating signal” to activate the transfer process. The products of traI and traM were also required both for donor conjugal DNA synthesis and for physical transfer of plasmid DNA, probably being involved in the conversion of covalently closed circular plasmid DNA into the open circular form that is the substrate for the independent although normally simultaneous synthesis and transfer steps. In contrast, donor conjugal DNA synthesis took place at a normal rate in both piliated traG and traN mutants, and at a reduced rate in traD mutants, although in no case was there physical transfer of plasmid DNA. These gene products are therefore required for DNA transfer to the recipient, and in addition, the absence of the traD product may hinder DNA synthesis.Based upon these results, a scheme for the processing of DNA during conjugation is presented.     

IR and UV studies on stability and conformations of short DNA duplexes containing a no-base residue: coexistence of B and Z conformations

Tridecamers containing a central no-base residue (X) have been synthesized and hybridized to their complementary strands, so as to constitute duplexes consisting of two hexamers separated by central mismatched X-A or X-T pairs. The effect of the introduction of this deoxyribose derivative on duplex stability was investigated by measuring UV absorbance as a function of salt concentration and temperature. As expected, the duplexes containing the abnormal base pairs (X-T and X-A) are less stable when compared to the totally complementary duplexes (A-T and T-A). The X-T mismatched duplex shows the most unstable thermodynamical behaviour. The conformational changes of these duplexes were studied by IR spectroscopy in condensed phase as a function of water content. At high relative humidity, the IR spectra show that these tridecamers form B-type double stranded duplex structures. If the water content is decreased, only the duplexes m5CGm5CGCTXAGCTTC GCGCGAATCGAAG and, to a lesser degree, m5CGm5CGCTXAGCTTC GCGGCATTCGAAG undergo a partial B---Z transition involving the methylated hexamer, the conformation of the second segment remaining of the B type. These results show that only one apurinic residue leads to a flexible junction between B and Z forms in a short duplex containing 5-methyl-2'-deoxycytidines.