A series of mono-, bis- and trisvinyl-pyridinium triphenylamines (TP-py) has been synthesised and evaluated for its one- and two-photon absorption (2PA) induced-fluorescence properties under biological conditions. Interestingly, these compounds are only weakly fluorescent in water, whereas their fluorescence emissions are strongly restored (exaltation factors of 20-100) upon binding to double-stranded DNA. Additional measurements in glycerol indicate that the fluorescence increases are the result of immobilisation of the dyes in the DNA matrix, which inhibits rotational de-excitation modes. This particular feature is especially remarkable in the case of the bis and tris derivatives (TP-2 py, TP-3 py), which each display a high affinity (K(d) ~ microM) for dsDNA. TPIF measurements have shown that TP-2 py and TP-3 py each have a large 2PA cross section (delta up to 700 GM) both in glycerol and in the presence of DNA, which ranks them amongst the best 2PA biological fluorophores. Finally, one- and two-photon confocal imaging in cells revealed that these compounds perform red staining (lambda(em)=660-680 nm) of nuclear DNA with excellent contrast. The remarkable optical properties of the TP-py series, combined with their high photostability and their easy synthetic access, make these compounds extremely attractive for use in confocal and 2PA microscopy.
A versatile synthetic strategy to access a set of highly fluorescent pi-conjugated triphenylamines bearing a functional linker at various positions on one phenyl ring is described. These compounds were designed for large two-photon absorption (2PA) and in particular for labeling of biomolecules. The monoderivatized trisformylated or trisiodinated intermediates described herein allow introduction of a large variety of electron-withdrawing groups required for large 2PA as well as a panel of chemical functions suitable for coupling to biomolecules. The monoderivatized three-branched compounds and in particular the benzothiazole (TP-3Bz) series show remarkable linear (high extinction coefficients and high quantum yield) and nonlinear (high 2-photon cross sections) optical properties. Interestingly the presence of functional side chains does not disturb the two-photon absorption. Finally, monoderivatized two-branched derivatives also appear to be valuable candidates. Altogether the good optical properties of the new derivatizable pi-conjugated TPA combined with their small size and their compatibility with bioconjugation protocols suggest that they represent a new chemical class of labels potentially applicable for the tracking of biomolecules using two-photon scanning microscopy.
A new concept for sequence‐specific labeling of DNA by using chemically modified cofactors for DNA methyltransferases is presented. Replacement of the amino acid side chain of the natural cofactor S‐adenosyl‐L‐methionine with an aziridine group leads to a cofactor suitable for DNA methyltransferase‐catalyzed sequence‐specific coupling with DNA. Sequence‐specifically fluorescently labeled plasmid DNA was obtained by using the DNA methyltransferase from Thermus aquaticus (M.TaqI) as catalyst and attaching a fluorophore to the aziridine cofactor. First results suggest that all classes of DNA methyltransferases with different recognition sequences can be used. In addition, this novel method for DNA labeling should be applicable to a wide variety of reporter groups.
A method to position nanoparticles onto DNA with high resolution using an enzyme-based approach is described. This provides a convenient route to assemble multiple nanoparticles (e.g., Au and CdSe) to specific positions with a high level of control and expandability to more complex assemblies. Atomic force microscopy is used to analyze the nanostructures, which have potential interest for biosensor, optical waveguide, molecular electronics, and energy transfer studies.
Sequence-specific labeling of native deoxyribonucleic acid (DNA) still represents a more-or-less unsolved problem. Difficulties mainly arise from the necessity to combine two different functions: sequence-specific recognition of DNA and covalent bond formation between the label and DNA. DNA methyltransferases (MTases) naturally possess these two functions and transfer a methyl group from the cofactor S-adenosyl-L-methionine (AdoMet) to adenine or cytosine residues within specific DNA sequences, typically ranging from two to eight base pairs. Unfortunately, the methyl group itself is a very limited reporter group and it would be desirable to transfer larger chemical entities with DNA MTases. Replacement of the methionine side chain of the natural cofactor AdoMet by an aziridinyl residue leads to the synthetic cofactor N-adenosylaziridine, which is quantitatively, base- and sequence-specifically coupled with DNA in a DNA MTase-catalyzed reaction. By attaching interesting reporter groups to a suitable position of N-adenosylaziridine a large variety of new synthetic cofactors are obtained for sequence-specific labeling of DNA. This method is illustrated by coupling primary amino groups and biotin to short duplex oligodeoxynucleotides or plasmid DNA using the DNA MTase M.TaqI.
Biomolecular self-assembly provides a basis for the bottom-up construction of useful and diverse nanoscale architectures. DNA is commonly used to create these assemblies and is often exploited as a lattice or an array. Although geometrically rigid and highly predictable, these sheets of repetitive constructs often lack the ability to be enzymatically manipulated or elongated by standard biochemical techniques. Here, we describe two approaches for the construction of position-controlled, molecular-scale, discrete, three- and four-way DNA junctions. The first approach for constructing these junctions relies on the use of nonmigrating cruciforms generated from synthetic oligonucleotides to which large, biologically generated, double-stranded DNA segments are enzymatically ligated. The second approach utilitizes the DNA methyltransferase-based SMILing (sequence-specific methyltransferase-induced labeling of DNA) method to site-specifically incorporate a biotin within biologically derived DNA. Streptavidin is then used to form junctions between unique DNA strands. The resultant assemblies have precise and predetermined connections with lengths that can be varied by enzymatic or hybridization techniques, or geometrically controlled with standard DNA functionalization methods. These junctions are positioned with single nucleotide resolution on large, micrometer-length templates. Both approaches generate DNA assemblies which are fully compatible with standard recombinant methods and thus provide a novel basis for nanoengineering applications.
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