[Biophysics of the DNA molecule: a new trend]. / Biofizika molekuly DNK: odno iz novykh napravlenii.

Briefly discussed are experiments with single molecules, representing a novel trend in the biophysical study of DNA. The techniques of optical and magnetic tweezers whereby external force can be applied to individual DNA molecules were used to assess the structural transitions of the DNA double helix under such conditions. Discussed are the latest data on the dependence of the rate of complementary chain synthesis by DNA polymerase on the stretching of the template.

PNA beacons for duplex DNA.

We report here on the hybridization of peptide nucleic acid (PNA)-based molecular beacons (MB) directly to duplex DNA sites locally exposed by PNA openers. Two stemless PNA beacons were tested, both featuring the same recognition sequence and fluorophore-quencher pair (Fluorescein and DABCYL, respectively) but differing in arrangement of these groups and net electrostatic charge. It was found that one PNA beacon rapidly hybridized, with the aid of openers, to its complementary target within duplex DNA at ambient conditions via formation of a PD-like loop. In contrast, the other PNA beacon bound more slowly to preopened duplex DNA target and only at elevated temperatures, although it readily hybridized to single-stranded (ss) DNA target. Besides a higher selectivity of hybridization provided by site-specific PNA openers, we expect this approach to be very useful in those MB applications when denaturation of the duplex DNA analytes is unfavorable or undesirable. Furthermore, we show that PNA beacons are advantageous over DNA beacons for analyzing unpurified/nondeproteinized DNA samples. This feature of PNA beacons and our innovative hybridization strategy may find applications in emerging fluorescent DNA diagnostics.

Tuning DNA "strings": modulating the rate of DNA replication with mechanical tension.

Recent experiments have measured the rate of replication of DNA catalyzed by a single enzyme moving along a stretched template strand. The dependence on tension was interpreted as evidence that T7 and related DNA polymerases convert two (n = 2) or more single-stranded template bases to double helix geometry in the polymerization site during each catalytic cycle. However, we find structural data on the T7 enzyme--template complex indicate n = 1. We also present a model for the "tuning" of replication rate by mechanical tension. This model considers only local interactions in the neighborhood of the enzyme, unlike previous models that use stretching curves for the entire polymer chain. Our results, with n = 1, reconcile force-dependent replication rate studies with structural data on DNA polymerase complexes.

High-purity preparation of a large DNA dumbbell.

We report on the efficient biochemical synthesis of a large DNA dumbbell starting from a pair of short DNA hairpins with long single-stranded tails of arbitrary sequence. The DNA dumbbell is obtained by enzymatic ligation yielding a 94-bp duplex stem closed at both termini by single-stranded loops of 5 nt. Following ligation, all unligated precursors and monoligated by-products were multiply biotinylated via nick-translation or primer-extension or both. Thus, they could readily be removed from the DNA dumbbell preparation by a mild biomagnetic separation procedure. The closed conformation of the purified DNA dumbbell was verified by its altered gel mobility as compared with unligated or monoligated samples and by an exonuclease assay. Considering the promising therapeutic potential of DNA dumbbells, the developed biosynthetic approach could be used for high-purity preparation of longer, covalently closed DNA decoys.

Sequence-specific targeting of duplex DNA by peptide nucleic acids via triplex strand invasion.

Because of a set of exceptional chemical, physical, and biological properties, polyamide or peptide nucleic acids (PNAs) hold a distinctive position among various synthetic ligands designed for DNA-targeting purposes. Cationic pyrimidine PNAs (cpyPNAs) represent a special group of PNAs, which effectively form strand invasion triplexes with double-stranded DNA (dsDNA) also known as P-loops. Extraordinary stability of the invasion triplexes and high sequence specificity of their formation combined with local opening of the DNA double helix within the P-loops make these complexes very attractive for sequence-specific manipulation with dsDNA. Important for applications is the fact that the discrimination between correct and mismatched binding sites in dsDNA by cpyPNAs is a nonequilibrium, kinetically controlled process. Therefore, a careful choice of experimental conditions that are optimal for the kinetic discrimination of correct versus mismatched cpyPNA binding is crucial for sequence-specific recognition of dsDNA by cpyPNAs. The experimental and theoretical data presented make it possible to select those solution parameters and cpyPNA constructions that are most favorable for sequence specificity without compromising the affinity of dsDNA targeting.

Peptide nucleic acid-assisted topological labeling of duplex dna.

Peptide nucleic acids (PNAs) are a family of synthetic polyamide mimics of nucleic acids that offer a variety of applications. Pyrimidine bis-PNAs can be used for rational design of novel interlocked DNA nanostructures, earring labels, representing locked pseudorotaxanes or locked catenanes. These structures are created through DNA ligase-mediated catenation of duplex DNA with a circularized oligonucleotide tag at a designated DNA site. The assembly is performed via formation of the PD-loop consisting of a pair of bis-PNA openers and the probe oligonucleotide. The openers locally expose one of the two strands of duplex DNA for hybridizing the probe, whose termini are complementary to the displaced DNA strand. After hybridization, they are in juxtaposition and can subsequently be linked by DNA ligase. As a result, a true topological link forms at a precise position on the DNA double helix yielding locked, earring-like label. DNA topological labeling can be done both in solution and, for longer templates, within the agarose gel plug. Accordingly, highly localized DNA detection with rolling circle amplification of hybridization signal and effective micromanipulations with DNA duplexes become possible through precise spatial positioning of various ligands on the DNA scaffold.

An artificial primosome: design, function, and applications.

Double-stranded (ds) DNA is capable of the sequence-specific accommodation of an additional oligodeoxyribonucleotide strand by the peptide nucleic acid(PNA)-assisted formation of a so-called PD-loop. We demonstrate here that the PD-loop may function as an artificial primosome within linear, nonsupercoiled DNA duplexes. DNA polymerase with its strand displacement activity uses this construct to initiate the primer extension reaction at a designated dsDNA site. The primer is extended by several hundred nucleotides. The efficiency of dsDNA priming by the artificial primosome assembly is comparable to the single-stranded DNA priming used in various assays. The ability of the PD-loop structure to perform like an artificial primosome on linear dsDNA may find applications in biochemistry, molecular biology, and molecular biotechnology, as well as for DNA diagnostics. In particular, multiple labels can be incorporated into a chosen dsDNA site resulting in ultrasensitive direct quantification of specific sequences. Furthermore, nondenaturing dsDNA sequencing proceeds from the PD-loop. This approach opens the way to direct isothermal reading of the DNA sequence against a background of unrelated DNA, thereby eliminating the need for purification of the target DNA.

Sequence-specific protection of duplex DNA against restriction and methylation enzymes by pseudocomplementary PNAs.

A new generation of PNAs, so-called pseudocomplementary PNAs (pcPNAs), which are able to target the designated sites on duplex DNA with mixed sequence of purines and pyrimidines via double-duplex invasion mode, has recently been introduced. It has been demonstrated that appropriate pairs of decameric pcPNAs block an access of RNA polymerase to the corresponding promoter. Here, we show that this type of PNAs protects selected DNA sites containing all four nucleobases from the action of restriction enzymes and DNA methyltransferases. We have found that pcPNAs as short as octamers form stable and sequence-specific complexes with duplex DNA in a very salt-dependent manner. In accord with a strand-invasion mode of complex formation, the pcPNA binding proceeds much faster with supercoiled than with linear plasmids. The double-duplex invasion complexes selectively shield specific DNA sites from BclI restriction endonuclease and dam methylase. The pcPNA-assisted protection against enzymatic methylation is more efficient when the PNA-binding site embodies the methylase-recognition site rather than overlaps it. We conclude that pcPNAs may provide the robust tools allowing to sequence-specifically manipulate DNA duplexes in a virtually sequence-unrestricted manner.

An earring for the double helix: assembly of topological links comprising duplex DNA and a circular oligodeoxynucleotide.

Abstract Novel DNA nanostructures, locked pseudorotaxane and locked catenane were assembled through topological linkage of a double-stranded target to a circular oligodeoxyribonucleotide (cODN)(+). The formation of these supramolecular complexes occurs with remarkable sequence specificity and is accomplished via local opening of duplex DNA by a pair of homopyrimidine bis-PNAs. The obtained cODN label, resembling an earring, forms a true topological link with the linear or closed circular (cc) target DNA and occupies a fixed position along the double helix. The PNA directed assembly described here introduces PNA oligomers into the repertoire of DNA nanotechnological tools.

PNA openers as a tool for direct quantification of specific targets in duplex DNA.

We report a new approach for target quantification directly within DNA duplex. Our assay is based on the formation of a new biomolecular structure, the PD-loop. The approach takes advantage of a selective hybridization of a probe to double-stranded DNA (dsDNA), which is locally opened by a pair of bis-PNA oligomers. To optimize the technique, several experimental formats are tested with the use of PNA and oligonucleotide probes. The highest sensitivity is achieved when the hybridized probe is extended and multiply labeled with 125I-dCTP by DNA polymerase via strand displacement in the presence of single-strand binding (SSB) protein. In this case, the PNA-assisted probe hybridization combined with the method of multiphoton detection (MPD) allows to monitor sub-attomolar amounts of the HIV-1 target on the background of unrelated DNA at sub-nCi level of radioactivity. The developed robust methodology is highly discriminative to single mutations, thus being of practical use for DNA analysis.

Oligonucleotide dendrimers: stable nano-structures.

DNA dendrimers with two, three, six, nine or 27 arms were reassociated as complementary pairs in solution or with an array of complementary oligonucleotides on a solid support. In all cases, duplex stabilities were greater than those of unbranched molecules of equal length. A theoretical treatment for the process of dissociation of dendrimers explains the major properties of the complexes. The favourable features of DNA dendrimers-their enhanced stability and the simple predictability of their association behaviour-makes them promising as building blocks for the 'bottom up' approach to nano-assembly. These features also suggest applications in oligonucleotide array/DNA chip technology when higher hybridisation temperatures are required, for example, to melt secon-dary structure in the target.

An experimental study of mechanism and specificity of peptide nucleic acid (PNA) binding to duplex DNA.

We investigated the mechanism and kinetic specificity of binding of peptide nucleic acid clamps (bis-PNAs) to double-stranded DNA (dsDNA). Kinetic specificity is defined as a ratio of initial rates of PNA binding to matched and mismatched targets on dsDNA. Bis-PNAs consist of two homopyrimidine PNA oligomers connected by a flexible linker. While complexing with dsDNA, they are known to form P-loops, which consist of a [PNA]2-DNA triplex and the displaced DNA strand. We report here a very strong pH-dependence, within the neutral pH range, of binding rates and kinetic specificity for a bis-PNA consisting of only C and T bases. The specificity of binding reaches a very sharp and high maximum at pH 6.9. In contrast, if all the cytosine bases in one of the two PNA oligomers within the bis-PNA are replaced by pseudoisocytosine bases (J bases), which do not require protonation to form triplexes, a weak dependence on pH of the rates and specificity of the P-loop formation is observed. A theoretical analysis of the data suggests that for (C+T)-containing bis-PNA the first, intermediate step of PNA binding to dsDNA occurs via Hoogsteen pairing between the duplex target and one oligomer of bis-PNA. After that, the strand invasion occurs via Watson-Crick pairing between the second bis-PNA oligomer and the homopurine strand of the target DNA, thus resulting in the ultimate formation of the P-loop. The data for the (C/J+T)-containing bis-PNA show that its high affinity to dsDNA at neutral pH does not seriously compromise the kinetic specificity of binding. These findings support the earlier expectation that (C/J+T)-containing PNA constructions may be advantageous for use in vivo.

Yeast artificial chromosome segregation from host chromosomes with similar lengths.

We propose a new method for segregation of yeast artificial chromosomes (YACs) from endogenous yeast chromosomes with similar lengths. The method is based on recently developed PNA-assisted rare cleavage (PARC) of genomic DNA. We apply the PARC procedure to YAC-containing samples of yeast DNA in such a way that host chromosomes, which electrophoretically comigrate with the chosen YACs, are selectively digested while YACs remain intact. These data demonstrate that a pool of appropriate PNAs can be used as an efficient tool for the PARC-based isolation of intact purified YACs directly from the host cells.

PD-loop: a complex of duplex DNA with an oligonucleotide.

A stable complex between duplex DNA and an oligonucleotide is assembled with the aid of a DNA synthetic mimic, peptide nucleic acid (PNA). Homopyrimidine PNAs are known to invade into short homopurine tracts in duplex DNA forming P-loops. We have found that P-loops, formed at two closely located purine tracts in the same DNA strand separated by a mixed purine-pyrimidine sequence, merge and open the double helix between them. The opposite DNA strand, which is not bound with PNA, exposes and becomes accessible for complexing with an oligonucleotide via Watson-Crick pairing. As a result, the PD-loop emerges, which consists of locally open duplex DNA, PNA "openers," and an oligonucleotide. The PD-loop stability and sequence specificity are demonstrated by affinity capture of duplex DNAs by using biotinylated oligonucleotides and streptavidin-covered magnetic beads. The type of complex formed by PNAs, an oligonucleotide and duplex DNA we describe, opens ways for development of various in vitro and in situ hybridization techniques with duplex DNA and may find applications in DNA nanotechnology and genomics.

A theoretical analysis of specificity of nucleic acid interactions with oligonucleotides and peptide nucleic acids (PNAs).

We treat theoretically the problem of the specificity of interaction between nucleic acid and an oligonucleotide, its analog or its mimic (such as peptide nucleic acid, or PNA). We consider simplest models with only essential details using numerical solutions of kinetic equations and the kinetic Monte Carlo method. In our first model, describing the formation of complementary duplex, we demonstrate anti-correlation between specificity and affinity for nucleic acid/oligonucleotide interaction. We analyze in detail one notable exception. Homopyrimidine PNAs exhibit very high affinity to DNA forming extraordinarily stable DNA/(PNA)2 triplexes with the complementary DNA strand. At the same time, such PNAs show remarkable sequence specificity of binding to duplex DNA. We formulate a theoretical model for the two-step process of PNA interaction with DNA. The calculations demonstrate that two-stage binding may secure both high affinity and very high specificity of PNA interaction with DNA. Our computer simulations define the range of parameter values in which high specificity is achieved. These findings are of great importance for numerous applications of PNA and for design of future drugs which specifically interact with DNA.

Kinetic sequence discrimination of cationic bis-PNAs upon targeting of double-stranded DNA.

Strand displacement binding kinetics of cationic pseudoisocytosine-containing linked homopyrimidine peptide nucleic acids (bis-PNAs) to fully matched and singly mismatched decapurine targets in double-stranded DNA (dsDNA) are reported. PNA-dsDNA complex formation was monitored by gel mobility shift assay and pseudo-first order kinetics of binding was obeyed in all cases studied. The kinetic specificity of PNA binding to dsDNA, defined as the ratio of the initial rates of binding to matched and mismatched targets, increases with increasing ionic strength, whereas the apparent rate constant for bis-PNA-dsDNA complex formation decreases exponentially. Surprisingly, at very low ionic strength two equally charged bis-PNAs which have the same sequence of nucleobases but different linkers and consequently different locations of three positive charges differ in their specificity of binding by one order of magnitude. Under appropriate experimental conditions the kinetic specificity for bis-PNA targeting of dsDNA is as high as 300. Thus multiply charged cationic bis-PNAs containing pseudoisocytosines (J bases) in the Hoogsteen strand combined with enhanced binding affinity also exhibit very high sequence specificity, thereby making such reagents extremely efficient for sequence-specific targeting of duplex DNA.

Kinetic analysis of specificity of duplex DNA targeting by homopyrimidine peptide nucleic acids.

A simple theoretical analysis shows that specificity of double-stranded DNA (dsDNA) targeting by homopyrimidine peptide nucleic acids (hpyPNAs) is a kinetically controlled phenomenon. Our computations give the optimum conditions for sequence-specific targeting of dsDNA by hpyPNAs. The analysis shows that, in agreement with the available experimental data, kinetic factors play a crucial role in the selective targeting of dsDNA by hpyPNAs. The selectivity may be completely lost if PNA concentration is too high and/or during prolonged incubation of dsDNA with PNA. However, quantitative estimations show that the experimentally observed differences in the kinetic constants for hpyPNA binding with the correct and mismatched DNA sites are sufficient for sequence-specific targeting of long genomic DNA by hpyPNAs with a high yield under appropriate experimental conditions. Differential dissociation of hpyPNA/dsDNA complexes is shown to enhance the selectivity of DNA targeting by PNA.

Theoretical treatment of melting of complexes of DNA with ligands having several types of binding sites on helical and single-stranded DNA.

We treat theoretically conformational transitions in DNA-ligand complexes allowing for the existence of different binding parameters of the ligand to different DNA conformations. The parameters of binding are determined from the best fit of the theory to experimental data for the difference between transition point (Tm) and the width of transition curve (delta T) for the complexes and for naked DNA. The analysis shows that Ethidium Bromide (EB) and Actinomycin D (AMD) each may form at least five types of complexes: three types (one "strong" and two "weak") with helix DNA and two types ("strong" and "weak") with single-stranded DNA. The parameters of the complexes have been obtained. Some testable experimental predictions of the theory are also discussed.

A new class of genome rare cutters.

Although significant efforts have been directed at developing efficient techniques for rare and super rare genome cutting, only limited success has been achieved. Here we propose a new approach to solve this problem. We demonstrate that peptide nucleic acid 'clamps' (bis-PNAs) bind strongly and sequence specifically to short homopyrimidine sites on lambda and yeast genomic DNAs. Such binding efficiently shields methylation/restriction sites which overlap with the bis-PNA binding sites from enzymatic methylation. After removing the bis-PNA, the genomic DNAs are quantitatively cleaved by restriction enzymes into a limited number of pieces of lengths from several hundred kbp to several Mbp. By combining various bis-PNAs with different methylation/restriction enzyme pairs, a huge new class of genome rare cutters can be created. These cutters cover the range of recognition specificities where very few, if any, cutters are now available.