Heiko Kuhn

Nanopore based sequence specific detection of duplex DNA for genomic profiling.

We demonstrate a purely electrical method for the single-molecule detection of specific DNA sequences, achieved by hybridizing double-stranded DNA (dsDNA) with peptide nucleic acid (PNA) probes and electrophoretically threading the DNA through sub-5 nm silicon nitride pores. Bis-PNAs were used as the tagging probes in order to achieve high affinity and sequence specificity. Sequence detection is performed by reading the ion current traces of individual translocating DNA molecules, which display a characteristic secondary blockade level, absent in untagged molecules. The potential for barcoding DNA is demonstrated through nanopore analysis of once-tagged and twice-tagged DNA at different locations on the same genomic fragment. Our high-throughput, long-read length method can be used to identify key sequences embedded in individual DNA molecules, without the need for amplification or fluorescent/radio labeling. This opens up a wide range of possibilities in human genomics as well as in pathogen detection for fighting infectious diseases.

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.

Template-independent ligation of single-stranded DNA by T4 DNA ligase

T4 DNA ligase is one of the workhorses of molecular biology and used in various biotechnological applications. Here we report that this ligase, unlike Escherichia coli DNA ligase, Taq DNA ligase and Ampligase, is able to join the ends of single‐stranded DNA in the absence of any duplex DNA structure at the ligation site. Such nontemplated ligation of DNA oligomers catalyzed by T4 DNA ligase occurs with a very low yield, as assessed by quantitative competitive PCR, between 10−6 and 10−4 at oligonucleotide concentrations in the range 0.1–10 nm, and thus is insignificant in many molecular biological applications of T4 DNA ligase. However, this side reaction may be of paramount importance for diagnostic detection methods that rely on template‐dependent or target‐dependent DNA probe ligation in combination with amplification techniques, such as PCR or rolling‐circle amplification, because it can lead to nonspecific background signals or false positives. Comparison of ligation yields obtained with substrates differing in their strandedness at the terminal segments involved in ligation shows that an acceptor duplex DNA segment bearing a 3′‐hydroxy end, but lacking a 5′‐phosphate end, is sufficient to play a role as a cofactor in blunt‐end ligation.

Topological Links between Duplex DNA and a Circular DNA Single Strand

DNA nanostructures of building blocks topologically linked at a precise position can be assembled from DNA duplexes and circularized oligonucleotides with the aid of peptide nucleic acids (PNAs). Shown schematically is a linked catenane yielding an earring topological DNA label.

Labeling of unique sequences in double-stranded DNA at sites of vicinal nicks generated by nicking endonucleases

We describe a new approach for labeling of unique sequences within dsDNA under nondenaturing conditions. The method is based on the site-specific formation of vicinal nicks, which are created by nicking endonucleases (NEases) at specified DNA sites on the same strand within dsDNA. The oligomeric segment flanked by both nicks is then substituted, in a strand displacement reaction, by an oligonucleotide probe that becomes covalently attached to the target site upon subsequent ligation. Monitoring probe hybridization and ligation reactions by electrophoretic mobility retardation assay, we show that selected target sites can be quantitatively labeled with excellent sequence specificity. In these experiments, predominantly probes carrying a target-independent 3′ terminal sequence were employed. At target labeling, thus a branched DNA structure known as 3′-flap DNA is obtained. The single-stranded terminus in 3′-flap DNA is then utilized to prime the replication of an externally supplied ssDNA circle in a rolling circle amplification (RCA) reaction. In model experiments with samples comprised of genomic λ-DNA and human herpes virus 6 type B (HHV-6B) DNA, we have used our labeling method in combination with surface RCA as reporter system to achieve both high sequence specificity of dsDNA targeting and high sensitivity of detection. The method can find applications in sensitive and specific detection of viral duplex DNA.

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.

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.

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.

Monitoring of single nicks in duplex DNA by gel electrophoretic mobility-shift assay.

We demonstrate that the gel electrophoretic mobility‐shift assay (EMSA) can be used for site‐selective and quantitative monitoring of nicks in linear double‐stranded DNA (dsDNA) thus allowing to expediently follow the nicking activity of enzymes or other agents targeted to a designated dsDNA site. At elevated temperature and/or in the presence of urea, DNA fragments carrying a single nick produced by the nicking enzyme N.BstNBI exhibit a well‐detectable gel retardation effect. On the basis of permutation analysis, the decreased electrophoretic mobility of nicked dsDNA fragments is attributed to a bend (or hinge) in the DNA double helix sequence‐specifically generated by a nick. Since nick‐induced DNA bending depends on interaction between base pairs adjacent to a nick, the change in mobility is different for nicked DNA sites with different sequences. Therefore, EMSA monitoring of differential mobility change caused by nicks within various DNA sequences could be useful for studying the differential base stacking and nearest‐neighbor energetics.

SYNTHESIS OF SEVERAL NOVEL OPTICALLY ACTIVE NITROXYL RADICALS

Abstract Optically active nitroxyl radicals are prepared from enantiomerically pure bicyclic terpenoids. (−)-Camphoxyl radical (−)-4 derived from commercially available oxazolidinone ( (−)-1 ) and (+)-camphoxyl radical (+)-4 derived from (−)-camphene are readily prepared, conformationally rigid, enantiomeric nitroxyl radicals. (−)-Camphorsulphonic acid is used to prepare two additional optically active nitroxyl radicals 9 and 12 .