ABEL-FRET: tether-free single-molecule FRET with hydrodynamic profiling.

Single-molecule Förster resonance energy transfer (smFRET) has become a versatile and widespread method to probe nanoscale conformation and dynamics. However, current experimental modalities often resort to molecule immobilization for long observation times and do not always approach the resolution limit of FRET-based nanoscale metrology. Here we present ABEL-FRET, an immobilization-free platform for smFRET measurements with ultrahigh resolving power in FRET efficiency. Importantly, single-molecule diffusivity is used to provide additional size and shape information for hydrodynamic profiling of individual molecules, which, together with the concurrently measured intramolecular conformation through FRET, enables a holistic and dynamic view of biomolecules and their complexes.

DNA-RNA Heteroduplex Oligonucleotide for Highly Efficient Gene Silencing.

Heteroduplex oligonucleotides (HDOs) were a novel type of nucleic acid drugs based on an antisense oligonucleotide (ASO) strand and its complementary RNA (cRNA ) strand. HDOs were originally designed to improve the properties of RNase H-dependent ASOs and we reported in our first paper that HDOs conjugated with an α-tocopherol ligand (Toc-HDO ) based on a gapmer ASO showed 20 times higher silencing effect to liver apolipoprotein B (apoB) mRNA in vivo than the parent ASO. Thereafter the HDO strategy was found to be also effective for improving the properties of ASOs modulating blood-brain barrier function and ASO antimiRs which are RNase H-independent ASOs. Therefore, the HDO strategy has been shown to be versatile technology platform to develop effective nucleic acid drugs.

Structural insights into complete metal ion coordination from ternary complexes of B family RB69 DNA polymerase.

We have captured a preinsertion ternary complex of RB69 DNA polymerase (RB69pol) containing the 3' hydroxyl group at the terminus of an extendable primer (ptO3') and a nonhydrolyzable 2'-deoxyuridine 5'-α,ß-substituted triphosphate, dUpXpp, where X is either NH or CH(2), opposite a complementary templating dA nucleotide residue. Here we report four structures of these complexes formed by three different RB69pol variants with catalytically inert Ca(2+) and four other structures with catalytically competent Mn(2+) or Mg(2+). These structures provide new insights into why the complete divalent metal-ion coordination complexes at the A and B sites are required for nucleotidyl transfer. They show that the metal ion in the A site brings ptO3' close to the α-phosphorus atom (Pα) of the incoming dNTP to enable phosphodiester bond formation through simultaneous coordination of both ptO3' and the nonbridging Sp oxygen of the dNTP's α-phosphate. The coordination bond length of metal ion A as well as its ionic radius determines how close ptO3' can approach Pα. These variables are expected to affect the rate of bond formation. The metal ion in the B site brings the pyrophosphate product close enough to Pα to enable pyrophosphorolysis and assist in the departure of the pyrophosphate. In these dUpXpp-containing complexes, ptO3' occupies the vertex of a distorted metal ion A coordination octahedron. When ptO3' is placed at the vertex of an undistorted, idealized metal ion A octahedron, it is within bond formation distance to Pα. This geometric relationship appears to be conserved among DNA polymerases of known structure.

Mechanism of homologous recombination from the RecA-ssDNA/dsDNA structures.

The RecA family of ATPases mediates homologous recombination, a reaction essential for maintaining genomic integrity and for generating genetic diversity. RecA, ATP and single-stranded DNA (ssDNA) form a helical filament that binds to double-stranded DNA (dsDNA), searches for homology, and then catalyses the exchange of the complementary strand, producing a new heteroduplex. Here we have solved the crystal structures of the Escherichia coli RecA-ssDNA and RecA-heteroduplex filaments. They show that ssDNA and ATP bind to RecA-RecA interfaces cooperatively, explaining the ATP dependency of DNA binding. The ATP gamma-phosphate is sensed across the RecA-RecA interface by two lysine residues that also stimulate ATP hydrolysis, providing a mechanism for DNA release. The DNA is underwound and stretched globally, but locally it adopts a B-DNA-like conformation that restricts the homology search to Watson-Crick-type base pairing. The complementary strand interacts primarily through base pairing, making heteroduplex formation strictly dependent on complementarity. The underwound, stretched filament conformation probably evolved to destabilize the donor duplex, freeing the complementary strand for homology sampling.

Stabilization of multi-stranded nucleic acid structures using 3'-S-phosphorothiolate linkages.

3 '-S-Phosphorothiolate linkages incorporated into an oligodeoxynucleotide have been shown to stabilise duplex formation with a complementary RNA strand, but destabilise a duplex formed with a complementary DNA strand. The four-stranded i-motif structure is also stabilised this modification.

Thermodynamic and kinetic characterization of duplex formation between 2'-O, 4'-C-methylene-modified oligoribonucleotides, DNA and RNA.

2'-O,4'-C-methylene-linked ribonucleotide derivatives, named LNA (locked nucleic acid) and BNA (bridged nucleic acid) are nucleic acid analogoues that have shown high-affinity recognition of DNA and RNA, and the employment of LNA oligomers for antisense activity, gene regulation and nucleic acid diagnostics seems promising. Here we show kinetic and thermodynamic results on the interaction of a series of 10 bases long LNA-DNA mixmers, gabmers as well as full length LNA's with the complementary DNA, RNA and LNA oligonucleotides in the presence and absence of 10 mM Mg(2+)- ions. Our results show no significant differences in the reaction thermodynamics and kinetics between the LNA species, only a tendency to stronger duplex formation with the gabmer and mixmer. Introduction of a few LNA's thus may be a better strategy, than using full length LNA's to obtain an oligonucleotide that markedly increases the strength of duplexes formed with the complementary DNA and RNA.

Effect of terminal amino acids on the stability and specificity of PNA-DNA hybridisation.

The effect of various charged or hydrophobic amino acids on the hybridisation of fully complementary and mismatch PNA-DNA duplexes was investigated via UV melting curve analysis. The results described here show that the thermal stability and binding specificity of PNA probes can be modified by conjugation to amino acids and these effects should be considered in experimental design when conjugating PNA sequences to solubility enhancing groups or cell transport peptides. Where stabilisation of a duplex is important, without there being a corresponding need for specific binding to fully complementary targets, the conjugation of multiple lysine residues to the C-terminus of PNA may be the best probe design. If, however, the key is to obtain maximum discrimination between fully complementary and mismatch targets, a replacement of glutamic acid for lysine as the routine solubility enhancing group is recommended.

Control of DNA conformation using 3'-S-phosphorothiolate-modified linkages.

An in-depth study into the incorporation of multiple 3-S-phosphorothiolate modifications into oligodeoxynucleotides (ODNs) and their subsequent effect on ODN/DNA and ODN/RNA duplex stability. 3-S-Phosphorothiolate linkages increase the stability of ODN/RNA duplexes and decrease the stability of ODN/DNA duplexes.

The effect of a single boranophosphate substitution with defined configuration on the thermal stability and conformation of a DNA duplex.

Substitution of one non-bridging oxygen in a natural phosphodiester internucleotide linkage with a borano (-BH3) group results in a chiral phosphorus center in boranophosphate. UV thermal melting profiles were recorded for DNA duplexes formed between a DNA 9-mer with either an Sp or a Rp boranophosphate linkage in the middle and the complementary DNA 9-mer, as well as for their unmodified parent duplex. The thermal stability of the DNA duplexes was in the order of normal > Sp borano > Rp borano. CD spectra of all three duplexes exhibited typical B-DNA profile, which closely resembled each other.

Studies on the structure and dynamics of the human telomeric G quadruplex by single-molecule fluorescence resonance energy transfer.

We have investigated the structure and unfolding kinetics of the human telomeric intramolecular G quadruplex by using single-molecule fluorescence resonance energy transfer. An exploration of conformational heterogeneity revealed two stable folded conformations, in both sodium- and potassium-containing buffers, with small differences between their enthalpies and entropies. Both folded conformations can be opened by the addition of a 21-base complementary DNA oligonucleotide. The unfolding of both substates occurs at the same rate, which showed dependence on the monovalent metal cation present. Temperature-dependence studies in 100 mM KCl gave an apparent activation enthalpy and entropy of 6.4 +/- 0.4 kcal.mol-1 and -52.3 +/- 1.4 cal.mol-1.K-1, respectively, indicating that the unfolding is entropically driven and can occur easily. In contrast, in 100 mM NaCl the respective values are 14.9 +/- 0.2 kcal.mol-1 and -23.0 +/- 0.8 cal.mol-1.K-1, suggesting a more significant enthalpic barrier. Molecular modeling suggests that the two species are likely to be the parallel and antiparallel quadruplex structures. The unfolding free energy barrier is estimated to be between 3 and 15 kBT based on Kramers' theory. We conclude that under near-physiological conditions these structures coexist and can interconvert on a minute time scale.

Kissing complex-mediated dimerisation of HIV-1 RNA: coupling extended duplex formation to ribozyme cleavage.

Initiation of retroviral genomic RNA dimerisation is mediated by the mutual interaction of the dimerisation initiation site (DIS) stem-loops near to the 5' end of the RNA. This process is thought to involve formation of a transient 'kissing' complex over the self-complementary loop bases, which then refolds into a more stable extended interaction. We have developed a novel experimental system that allows us to clearly detect the extended duplex in vitro. Ribozyme sequences were incorporated into or adjacent to the type 1 human immunodeficiency virus DIS stem, leading to the formation of a functional ribozyme only in the extended duplex conformer. Here we show that extended duplex formation results in ribozyme cleavage, thus demonstrating the double-stranded nature of the extended complex and confirming that refolding occurs via melting of the DIS stems. Loop complementarity is essential for extended duplex formation but no sequence requirements for the loops were observed. Efficiency of extended duplex formation is dependent on the strength of the loop-loop interaction, temperature, the magnesium concentration and is strongly accelerated by the viral nucleocapsid protein NCp7. Our ribozyme-coupled approach should be applicable to the analyses of other refolding processes involving RNA loop-loop interactions.

Insights into peptide nucleic acid (PNA) structural features: the crystal structure of a D-lysine-based chiral PNA-DNA duplex.

Peptide nucleic acids (PNAs) are oligonucleotide analogues in which the sugar-phosphate backbone has been replaced by a pseudopeptide skeleton. They bind DNA and RNA with high specificity and selectivity, leading to PNA-RNA and PNA-DNA hybrids more stable than the corresponding nucleic acid complexes. The binding affinity and selectivity of PNAs for nucleic acids can be modified by the introduction of stereogenic centers (such as D-Lys-based units) into the PNA backbone. To investigate the structural features of chiral PNAs, the structure of a PNA decamer containing three D-Lys-based monomers (namely H-GpnTpnApnGpnAdlTdlCdlApnCpnTpn-NH2, in which pn represents a pseudopeptide link and dl represents a D-Lys analogue) hybridized with its complementary antiparallel DNA has been solved at a 1.66-A resolution by means of a single-wavelength anomalous diffraction experiment on a brominated derivative. The D-Lys-based chiral PNA-DNA (LPD) heteroduplex adopts the so-called P-helix conformation. From the substantial similarity between the PNA conformation in LPD and the conformations observed in other PNA structures, it can be concluded that PNAs possess intrinsic conformational preferences for the P-helix, and that their flexibility is rather restricted. The conformational rigidity of PNAs is enhanced by the presence of the chiral centers, limiting the ability of PNA strands to adopt other conformations and, ultimately, increasing the selectivity in molecular recognition.

The synaptic complex of RecA protein participates in hybridization and inverse strand exchange reactions.

RecA protein catalyzes strand exchange between homologous single-stranded and double-stranded DNAs. In the presence of ATPgammaS, the post-strand exchange synaptic complex is a stable end product that can be studied. Here we ask whether such complexes can hybridize to or exchange with DNA, 2'-OMe RNA, PNA, or LNA oligonucleotides. Using a gel mobility shift assay, we show that the displaced strand of a 45 bp synaptic complex can hybridize to complementary oligonucleotides with different backbones to form a four-stranded (double D-loop) joint that survives removal of the RecA protein. This hybridization reaction, which confirms the single-stranded character of the displaced strand in a synaptic complex, might initiate recombination-dependent DNA replication if it occurs in vivo. We also show that either strand of the heteroduplex in a 30 bp synaptic complex can be replaced with a homologous DNA oligonucleotide in a strand exchange reaction that is mediated by the RecA filament. Consistent with the important role that deoxyribose plays in strand exchange, oligonucleotides with non-DNA backbones did not participate in this reaction. The hybridization and strand exchange reactions reported here demonstrate that short synaptic complexes are dynamic structures even in the presence of ATPgammaS.

Differential helix stabilities and sites pre-organized for tertiary interactions revealed by monitoring local nucleotide flexibility in the bI5 group I intron RNA.

The local environment at adenosine residues in the bI5 group I intron RNA was monitored as a function of Mg(2+) using both the traditional method of dimethyl sulfate (DMS) N1 methylation and a new approach, selective acylation of 2'-amine substituted nucleotides. These probes yield complementary structural information because N1 methylation reports accessibility at the base pairing face, whereas 2'-amine acylation scores overall residue flexibility. 2'-Amine acylation robustly detects RNA secondary structure and is sensitive to higher order interactions not monitored by DMS. Disruption of RNA structure due to the 2'-amine substitution is rare and can be compensated by stabilizing folding conditions. Peripheral helices that do not interact with other parts of the RNA are more stable than both base paired helices and tertiary interactions in the conserved catalytic core. The equilibrium state of the bI5 intron RNA, prior to assembly with its protein cofactor, thus features a relatively loosely packed core anchored by more stable external stem-loop structures. Adenosine residues in J4/5 and P9.0 form structures in which the nucleotide is constrained but the N1 position is accessible, consistent with pre-organization to form long-range interactions with the 5' and 3' splice sites.

Site-specific strand breaks in RNA produced by (125)I radiodecay.

Decay of (125)I produces a shower of low energy electrons (Auger electrons) that cause strand breaks in DNA in a distance-dependent manner with 90% of the breaks located within 10 bp from the decay site. We studied strand breaks in RNA molecules produced by decay of (125)I incorporated into complementary DNA oligonucleotides forming RNA/DNA duplexes with the target RNA. The frequencies and distribution of the breaks were unaffected by the presence of the free radical scavenger dimethyl sulfoxide (DMSO) or by freezing of the samples. Therefore, as was the case with DNA, most of the breaks in RNA were direct rather than caused by diffusible free radicals produced in water. The distribution of break frequencies at individual bases in RNA molecules is narrower, with a maximum shifted to the 3'-end with respect to the distribution of breaks in DNA molecules of the same sequence. This correlates with the distances from the radioiodine to the sugars of the corresponding bases in A-form (RNA/DNA duplex) and B-form (DNA/DNA duplex) DNA. Interestingly, when (125)I was located close to the end of the antisense DNA oligonucleotide, we observed breaks in RNA beyond the RNA/DNA duplex region. This was not the case for a control DNA/DNA hybrid of the same sequence. We assume that for the RNA there is an interaction between the RNA/DNA duplex region and the single-stranded RNA tail, and we propose a model for such an interaction. This report demonstrates that (125)I radioprobing of RNA could be a powerful method to study both local conformation and global folding of RNA molecules.

Mechanism of spontaneous DNA-DNA interaction of homologous linear duplexes.

Previously, we demonstrated the interaction of homologous linear duplexes with formation of four-way DNA structures on the model of five PCR products. We propose that homologous duplex interaction is initiated by the nucleation of several dissociated base pairs of the complementary ends of two fragments with Holliday junction formation, in which cross point migration occurs via spooling of DNA strands from one duplex to the other one, finally resulting in complete resolution into new or previously existing duplexes. To confirm that DNA-DNA interaction involves formation of four-way DNA structures with strand exchange at the cross point, we have demonstrated the strand exchange process between identical duplexes using homologous fragments, harboring either biotin label or (32)P-label. Incubation of the mixture resulted in the addition of (32)P-label to biotin-labeled fragments, and the intensity of (32)P-labeling of biotinylated fragments was dependent upon the incubation duration. DNA-DNA interaction is not based on surface-dependent denaturing, as Triton X-100 does not decrease the formation of complexes between DNA duplexes. The equilibrium concentration of Holliday junctions depends on the sequences of the fragment ends and the incubation temperature. The free energy of Holliday junction formation by the fragments with GC and AT ends differed by 0.6 kcal/mol. Electron microscopic analysis demonstrated that the majority of Holliday junctions harbor the cross point within a 300 base pair region of the fragment ends. This insight into the mechanism of homologous duplex interaction extends our understanding of different DNA rearrangements. Understanding of DNA-DNA interaction is of practical use for better interpretation and optimization of PCR-based analyses.

Conformational analysis of site-specific DNA cross-links of cisplatin-distamycin conjugates.

The requirement for novel platinum antitumor drugs led to the concept of synthesis of novel platinum drugs based on targeting cisplatin to various carrier molecules. We have shown [Loskotova, H., and Brabec, V. (1999) Eur. J. Biochem. 266, 392-402] that attachment of DNA minor-groove-binder distamycin to cisplatin changes several features of DNA-binding mode of the parent platinum drug. Major differences comprise different conformational changes in DNA and a considerably higher interstrand cross-linking efficiency. The studies of the present work have been directed to the analysis of oligodeoxyribonucleotide duplexes containing single, site-specific adducts of platinum-distamycin conjugates. These uniquely modified duplexes were analyzed by Maxam-Gilbert footprinting, phase-sensitive gel electrophoresis bending assay and chemical probes of DNA conformation. The results have indicated that the attachment of distamycin to cisplatin mainly affects the sites involved in the interstrand cross-links so that these adducts are preferentially formed between complementary guanine and cytosine residues. This interstrand cross-link bends the helix axis by approximately 35 degrees toward minor groove, unwinds DNA by approximately 95 degrees and distorts DNA symmetrically around the adduct. In addition, CD spectra of restriction fragments modified by the cisplatin-distamycin conjugates have demonstrated that distamycin moiety in the interstrand cross-links of these compounds interacts with DNA. This interaction facilitates the formation of these adducts. Hence, the structural impact of the specific interstrand cross-link detected in this study deserves attention when biological behavior of cisplatin derivatives targeted by oligopeptide DNA minor-groove-binders is evaluated.

Discrimination between translesion synthesis and template switching during bypass replication of thymine dimers in duplex DNA.

The goal of this study was to determine whether bypass replication occurs by translesion synthesis or template switching (copy choice) when a duplex molecule carrying a single cis,syn-cyclobutane thymine dimer is replicated in vitro by human cell extracts. Circular heteroduplex DNA molecules were constructed to contain the SV40 origin of replication and a mismatch opposite to or nearby the dimer. Control molecules with only the mismatch were also prepared. Heteroduplexes were methylated at CpG islands and replicated in vitro (30 min). Following bisulfite treatment, the nascent DNA complementary to the dimer-containing template was distinguished from the other three strands by methylation-specific polymerase chain reaction. Cloning and sequencing of polymerase chain reaction products revealed that 80-98% carried the sequence predicted for translesion synthesis, with two adenines incorporated opposite the dimer. The fraction of clones with sequence predictive of template switching was reduced when extracts deficient in mismatch repair or nucleotide excision repair activities were used to replicate the heteroduplex molecules. These results support the conclusion that lesion bypass during in vitro replication of duplex DNA containing thymine dimers occurs by translesion synthesis.

Selectivity of polyamines on the stability of RNA-DNA hybrids containing phosphodiester and phosphorothioate oligodeoxyribonucleotides.

RNA-DNA hybrid stabilization is an important factor in the efficacy of oligonucleotide-based antisense gene therapy. We studied the ability of natural polyamines, putrescine, spermidine, and spermine, and a series of their structural analogues to stabilize RNA-DNA hybrids using melting temperature (Tm) measurements, circular dichroism (CD) spectroscopy, and the ethidium bromide (EB) displacement assay. Phosphodiester (PO) and phosphorothioate (PS) oligodeoxyribonucleotides (ODNs) (21-mer) targeted to the initiation codon region of c-myc mRNA and the corresponding complementary RNA oligomer were used for this study. In the absence of polyamines, the Tm values of RNA-PODNA and RNA-PSDNA helices were 41 +/- 1 and 35 +/- 1 degrees C, respectively, in 10 mM sodium cacodylate buffer. In the presence of a hexamine analogue of spermine at a concentration of 25 microM, the hybrids were stabilized with Tm values of 80 and 78 degrees C, for RNA-PODNA and RNA-PSDNA, respectively. The d(Tm)/d(log[polyamine]) values, representing the concentration-dependent stabilization of hybrid helices by polyamines, increased from 10 to 24 for both the RNA-PODNA and RNA-PSDNA helices. Bisethyl substitution of the primary amino groups of the polyamines reduced the hybrid stabilizing potential of the polyamines. Among the homologues of spermidine [H2N(CH2)3NH(CH2)nNH2, where n = 2-8; n = 4 for spermidine] and spermine [H)N(CH2)3NH(CH2)nNH(CH2)3NH2, where n = 2-8; n = 4 for spermine], spermidine and spermine were the most effective agents for stabilizing the hybrid helices. At a physiologically compatible concentration of 150 mM NaCl, the hybrid helix formed from PODNA was more stable than that formed from PSDNA in the presence of polyamines. CD spectroscopic studies showed that the hybrids were stabilized in a conformation close to A-DNA in the presence of polyamines. The relative binding affinity of the polyamine homologues for the hybrid helices, as measured by the EB displacement assay, followed the same order in which they stabilized the hybrids. These results are important in the antisense context and in the general context of polyamine-nucleic acid interactions, and suggest that pentamine and hexamine analogues of spermine might be useful in improving the efficacy of therapeutic ODNs.

Solution structure of the (+)-cis-anti-benzo[a]pyrene-dA ([BP]dA) adduct opposite dT in a DNA duplex.

Minor adducts, derived from the covalent binding of anti-benzo[a]pyrene-7,8-dihydroxy-9,10-epoxide to cellular DNA, may play an important role in generating mutations and initiating cancer. We have applied a combined NMR-computational approach including intensity based refinement to determine the solution structure of the minor (+)-cis-anti-[BP]dA adduct positioned opposite dT in the d(C1-T2-C3-T4-C5-[BP]A6-C7-T8-T9-C10-C11). (d(G12-G13-A14-A15-G16-T17-G18-A19-G20+ ++-A21-G22) 11-mer duplex. The BP ring system is intercalated toward the 5'-side of the [BP]dA6 lesion site without disrupting the flanking Watson-Crick dC5.dG18 and [BP]dA6.dT17 base pairs. This structure of the (+)-cis-anti-[BP]dA.dT 11-mer duplex, containing a bay region benzo[a]pyrenyl [BP]dA adduct, is compared with the corresponding structure of the (+)-trans-anti-[BPh]dA.dT 11-mer duplex (Cosman et al., Biochemistry 32, 12488-12497, 1993), which contains a fjord region benzo[c]phenanthrenyl [BPh]dA adduct with the same R stereochemistry at the linkage site. The carcinogen intercalates toward the 5'-direction of the modified strand in both duplexes (the adduct is embedded within the same sequence context) with the buckling of the Watson-Crick [BP]dA6.dT17 base pair more pronounced in the (+)-cis-anti-[BP]dA.dT 11-mer duplex compared to its Watson-Crick [BPh]dA.dT17 base pair in the (+)-trans-anti-[BPh]dA.dT 11-mer duplex. The available structural studies of covalent polycyclic aromatic hydrocarbon (PAH) carcinogen-DNA adducts point toward the emergence of a general theme where distinct alignments are adopted by PAH adducts covalently linked to the N(6) of adenine when compared to the N(2) of guanine in DNA duplexes. The [BPh]dA and [BP]dA N(6)-adenine adducts intercalate their polycyclic aromatic rings into the helix without disruption of their modified base pairs. This may reflect the potential flexibility associated with the positioning of the covalent tether and the benzylic ring of the carcinogen in the sterically spacious major groove. By contrast, such an intercalation without modified base pair disruption option appears not to be available to [BP]dG N(2)-guanine adducts where the covalent tether and the benzylic ring are positioned in the more sterically crowded minor groove. In the case of [BP]dG adducts, the benzopyrenyl ring is either positioned in the minor groove without base pair disruption, or if intercalated into the helix, requires disruption of the modified base pair and displacement of the bases out of the helix.