Complete sequencing of mono—deprotonated peptide nucleic acids by sustained off-resonance irradiation collision—induced dissociation

Complete peptide nucleic acids (PNAs) sequence information is obtained from the unimolecular decomposition of singly-charged PNA oligomers in the negative-ion mode using electrospray ionization coupled with Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) and sustained off-resonance irradiation collision induced dissociation. The 4-mers, n-CATT-c, n-AGCT-c, n-AACT-c, and n-acetylated-AACT-c and two 6-mers, n-AAAAAA-c and n-CCCCCC-c, were investigated to explore the unimolecular decomposition of mixed-nucleobase and homopolymer PNAs representing purine and pyrimidine oligomers, respectively. PNA decomposition is explored using a product-ion appearance curve and double resonance experiments. A decomposition mechanism for sequence ion formation (PNA amide bond cleavage) is proposed.     

One-step, regioselective synthesis of up to 50-mers of RNA oligomers by montmorillonite catalysis

5'-Nucleotides of A and U with the phosphate activated with 1-methyladenine generate RNA oligomers containing 40-50 monomers in 1 day in reactions catalyzed by montmorillonite. The corresponding monomers of C give oligomers that are 20-25-mers in length after a 9-day reaction. It was not possible to determine the chain lengths of the oligomers of G since they did not give well-defined bands on gel electrophoresis. Co-oligomers of A and U as well as A, U, G, and C were also prepared. The oligo(A)s formed were separated by gel electrophoresis, and the bands of the 7-39-mers were isolated, the 3',5'-phosphodiester bonds were cleaved by RNase T(2), and the terminal phosphate groups were cleaved with alkaline phosphatase. HPLC analysis revealed that the proportions of A(5)'pp(5)'A, A, A(2)'pA, and A(2)'pA(2)'pA formed were almost the same for the long and shorter oligomers. A similar structure analysis performed on the oligo(U)s established that the proportions of U(5)'pp(5)'U, U, U(2)'pU, U(2)'pU(2)'pU, U(2)'pU(2)'pU(2)'pU, and U(2)'pU(2)'pU(2)'pU(2)'pU did not vary with chain length. The structural analysis of the oligomers of A revealed that 74% of the phosphodiester bonds were 3',5'-linked a value slightly greater than 67% observed when imidazole was the activating group. 61% of the bonds in the U oligomers were 3',5'-linked, which is almost 3 times greater than the 20% measured when imidazole was the activating group. The potential significance of these data to the origin and early evolution of life is discussed.     

In situ, on-resin synthesis of 8-Br/NH2 adeninyl peptide nucleic acid (PNA) oligomers and complementation studies with DNA

A new, novel and efficient in situ synthesis of 8-aminoadeninyl PNA oligomers from corresponding 8-bromoadeninyl PNA oligomers is reported. The study of hybridisation properties of (8-Br/8-NH₂) PNA oligomers with cDNA reveals substitution-site dependent stabilization of derived triplexes and duplexes.     

Hybridization Study of PNA-DNA in the Solution and Surface-Solution Interface for Biosensor Application

Abstract

Hybridization of 12-mers peptide nucleic acid (PNA) to complementary DNA was investigated in solution and on gold surfaces. The oligomers were designed to improve mismatch discrimination and minimize formation of secondary structures. Thermal denaturation experiments indicate high thermal stabilities for PNA-DNA hybrid with T _m values close to calculated values. Hybridization of PNA-DNA at 45°C and room temperature showed no difference. Hybridization on gold surface was also investigated with complementary and noncomplementary DNAs. The results show that 12-mer PNA and DNA hybridization at room temperature retained high specificity within ～5 ng.     

Label-free characterisation of oligonucleotide hybridisation using reflectometric interference spectroscopy

The potential of a label-free detection method, reflectometric interference spectroscopy (RIfS), for temperature-dependent DNA hybridisation experiments (for example in single nucleotide polymorphism (SNP) analysis) is investigated. Hybridisations of DNA, peptide nucleic acid (PNA), and locked nucleic acid (LNA) to a single stranded DNA were measured for several temperatures, and the melting curves and temperatures were calculated from the changes in optical thickness obtained. These measurements were performed by hybridising surface-immobilised single stranded oligomers with their complementary ssDNA or with ssDNA containing SNPs at different temperatures. DNA was compared to its analogue oligomers PNA and LNA due to their stability against nuclease. A comparison of melting temperatures demonstrated the higher binding affinities of the DNA analogues. Moreover, a continuous melting curve was obtained by first hybridising the functionalised surface with its complementary DNA at room temperature and then heating up in-flow. Measurement of the continuous melting curve was only possible due to the insensitivity of the RIfS method towards temperature changes. This is an advantage over other label-free detection methods, which are based on determining the refractive index.Dedicated to the memory of Wilhelm Fresenius.     

Sequence-specific binding of DNA to liposomes containing di-alkyl peptide nucleic acid (PNA) amphiphiles

We present a method to covalently attach peptide nucleic acid (PNA) to liposomes by conjugation of PNA peptide to charged amino acids and synthetic di-alkyl lipids ("PNA amphiphile," PNAA) followed by co-extrusion with disteroylphosphatidylcholine (DSPC) and cholesterol. Attachment of four Glu residues and two ethylene oxide spacers to the PNAA was required to confer proper hydration for extrusion and presentation for DNA hybridization. The extent of DNA oligomer binding to 10-mer PNAA liposomes was assessed using capillary zone electrophoresis. Nearly all PNAs on the liposome surface are complexed with a stoichiometric amount of complementary DNA 10-mers after 3-h incubation in pH 8.0 Tris buffer. No binding to PNAA liposomes was observed using DNA 10-mers with a single mismatch. Longer DNA showed a greatly attenuated binding efficiency, likely because of electrostatic repulsion between the PNAA liposome double layer and the DNA backbone. Langmuir isotherms of PNAA:DSPC:chol monolayers indicate miscibility of these components at the compositions used for liposome preparation. PNAA liposomes preserve the high sequence-selectivity of PNAs and emerge as a useful sequence tag for highly sensitive bioanalytical devices.     

Parallel synthesis of PNA-peptide conjugate libraries

An optimized semi-automatic protocol for parallel synthesis of up to 96 peptide nucleic acids (PNA) or PNA-peptide conjugates using Boc-protection strategy has been developed using a robotic system. The approach is illustrated by synthesizing PNA and PNA-peptide libraries varying between 15 and 27 amino acid units. The peptides (NLS (nuclear localization signal) or Tat-peptide) were attached to N-terminus of the PNA. The method was found to be far superior to that based on the SPOT/Fmoc protocol by which PNA oligomers are synthesized on a modified cellulose membrane. On a 0.5 micromole scale the method typically yielded 2 mg product of 90% purity by HPLC/MALDI-TOF analysis. This approach is suitable for screening of a large number of PNA and/or peptide sequences for biochemical and biological studies.     

Mass spectrometry characterization of Escherichia coli K4 oligosaccharides from 2-mers to more than 20-mers

The separation and characterization of oligosaccharides obtained by hyaluronidase [E.C. 3.2.1.35] digestion of Escherichia coli K4 polysaccharide using online high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) are presented. Complete identification and structural information for oligosaccharides containing 2-24 monomers (from 2- to 24-mers) were obtained. In particular, smaller K4 species, from 2-mers to 4-mers, exhibited mainly [M-H](-1) anions, whereas the 6- to 8-mers existed predominantly at the charge state of -2. The K4 oligomers from 10-mers to 14-mers were mainly represented by [M-3H](-3) anions while species from 16- to 20-mers were characterized by a charge state of -4. K4 oligosaccharides from 22- to 24-mers existed as [M-4H](-4) and [M-5H](-5) anions and, for this latter species, ions having a charge state of -6 appeared. For smaller K4 species, in particular from 6-mers to 10-mers, ESI-MS revealed anions related to the loss of one monosaccharide unit from the oligomers due to apparent collisional activation and ion source fragmentation. However, no odd-numbered anions were produced for K4 2/4-mer species or for oligosaccharides greater than 12-mers, while for K4 species 8/10-mer, ESI-MS revealed odd-numbered anions generally in low relative abundance making the interpretation of the spectra easier. The ESI-MS spectra of oligosaccharides separated by online HPLC were applied to the evaluation of the K4 polymerization process, confirming that the addition of fructose units is not critical for chain elongation as variously fructosylated oligomer species were detected directly on the K4 carbohydrate backbone.     

(1S,2R/1R,2S)-cis-cyclopentyl PNAs (cpPNAs) as constrained PNA analogues: synthesis and evaluation of aeg-cpPNA chimera and stereopreferences in hybridization with DNA/RNA

Conformationally constrained chiral PNA analogues were designed on the basis of stereospecific imposition of a 1,2-cis-cyclopentyl moiety on an aminoethyl segment of aegPNA. It is known that the cyclopentane ring is a relatively flexible system in which the characteristic puckering dictates the pseudoaxial/pseudoequatorial dispositions of substituents. Hence, favorable torsional adjustments are possible to attain the necessary hybridization-competent conformations when the moiety is imposed on the conventional PNA backbone. The synthesis of the enantiomerically pure 1,2-cis-cyclopentyl PNA monomers (10a and 10b) was achieved by stereoselective enzymatic hydrolysis of a key intermediate ester 2. The chiral (1S,2R/1R,2S)-aminocyclopentylglycyl thymine monomers were incorporated into PNA oligomers at defined positions and through the entire sequence. Hybridization studies with complementary DNA and RNA sequences using UV-Tm measurements indicate that aeg-cpPNA chimera form thermally more stable complexes than aegPNA with stereochemistry-dependent selective binding of cDNA/RNA. Differential gel shift retardation was observed on hybridization of aeg-cpPNAs with complementary DNA.     

Incorporation of guanosine gels into sieving matrices for length- and sequence-based separation of DNA in capillary electrophoresis

Sieving gels are used in capillary gel electrophoresis to resolve DNA strands of different lengths. For complex samples, however, such as those encountered in metagenomic analysis of microbial communities or biofilms, length-based separation may mask the true genetic diversity of the community since different organisms may contribute same-length DNA with different sequences. There is a need, therefore, for DNA separations based on both the length and sequence. Previous work has demonstrated the ability of guanosine gels (G-gels) to separate four single-stranded DNA 76-mers that differ by only a few A/G base substitutions. The goal of the present work is to determine whether G-gels could be combined with commercial sieving gels in order to simultaneously separate DNA based on both length and sequence. The results are given for the four 76-mers and for a standard dsDNA ladder. Commercial sieving gels were used alone and in combination with G-gels. For the 76-mers, the combined medium was less efficient than the G-gel alone but was able to achieve partial resolution. The combined medium was at least as effective as the sieving gel alone at resolving the denatured DNA ladder and showed indications of sequence-based resolution as well, as supported by MALDI-MS. The results show that the combined sieving gel/G-gel medium retains the selectivity of the individual media, providing a promising approach to simultaneous length- and sequence-based DNA separation for metagenomic analysis of complex systems.     

Sequential DNA hybridisation assays by fast micromixing

The prospects of performing DNA hybridisation assays in a novel sequential scheme are explored in this article. It is based on recording the kinetics of hybridisation on a microfluidic device measuring only 10 by 5 mm. It contains a split channel system for fast mixing and a subsequent meandering channel to observe the evolution of the mixture by optical means. The problems of diffusion limitations in the laminar flow regime are overcome by reducing the average diffusion distance to a few micrometers only. DNA oligomers (20-mers) of different sequences were injected on the chip for mixing. The detection of hybridisation was based on the fluorescence of DNA-intercalating dyes. Two modes of operation were investigated. First, the samples were injected into the micromixing device at a high flow rate of 40 microl min(-1). When the sample passed through the actual micromixing unit, the flow rate was reduced to allow for measurement of fluorescence levels at various steady-state reaction times in the range of 2-15 s, as defined by the channel geometry. Using this continuous flow approach, photobleaching of fluorophores could be avoided. In a buffer containing 0.2 M NaCl, 2 base-pair mismatches could routinely be detected within 5-20 s. Single base-pair mismatches were successfully identified under low salt conditions. In the second mode, the flow was completely stopped and the evolution of the total fluorescence signal influenced by the hybridisation of oligomers and photobleaching was observed. Whereas the sequence-dependent effects remained unchanged, the assay times between the mixing of two oligomers and clear identification of their hybridisation properties could be reduced down to a maximum of 5-7 s, in some cases even below 1 s.     

Molecularly defined caprolactone oligomers and polymers: synthesis and characterization

The synthesis of molecularly defined epsilon-caprolactone oligomers and polymers up to the 64-mer, via an exponential growth strategy, is described. By careful selection of orthogonal protecting groups, t-butyldimethylsilyl (TBDMS) ether for the hydroxyl group and benzyl (Bn) ester for the carboxylic acid group, a highly efficient synthetic strategy was developed with yields for both deprotection steps being essentially quantitative and for the coupling reactions using 1,3-dicyclohexylcarbodiimide (DCC), yields of 80-95% were obtained even at high molecular weights. This allows monodisperse dimers, tetramers, octamers, 16-mers, 32-mers and 64-mers to be prepared in gram quantities and fully characterized using mass spectroscopy, size exclusion chromatography (SEC), and IR and NMR spectroscopy. Thermal and physical properties were measured using thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), atomic force microscopy (AFM), and small-angle X-ray scattering (SAXS). These results conclusively show a distinct structure/property relationship with a close correlation between the number of repeat units and physical properties. In addition, a number of marked differences were observed on comparison with the parent poly(caprolactone) polymer.     

Electrochemiluminescent monomers for solid support syntheses of Ru(II)-PNA bioconjugates: multimodal biosensing tools with enhanced duplex stability

The feasibility of devising a solid support mediated approach to multimodal Ru(II)-peptide nucleic acid (PNA) oligomers is explored. Three Ru(II)-PNA-like monomers, [Ru(bpy)(2)(Cpp-L-PNA-OH)](2+) (M1), [Ru(phen)(2)(Cpp-L-PNA-OH)](2+) (M2), and [Ru(dppz)(2)(Cpp-L-PNA-OH)](2+) (M3) (bpy = 2,2'-bipyridine, phen = 1,10-phenanthroline, dppz = dipyrido[3,2-a:2',3'-c]phenazine, Cpp-L-PNA-OH = [2-(N-9-fluorenylmethoxycarbonyl)aminoethyl]-N-[6-(2-(pyridin-2yl)pyrimidine-4-carboxamido)hexanoyl]-glycine), have been synthesized as building blocks for Ru(II)-PNA oligomers and characterized by IR and (1)H NMR spectroscopy, mass spectrometry, electrochemistry and elemental analysis. As a proof of principle, M1 was incorporated on the solid phase within the PNA sequences H-g-c-a-a-t-a-a-a-a-Lys-NH(2) (PNA1) and H-P-K-K-K-R-K-V-g-c-a-a-t-a-a-a-a-lys-NH(2) (PNA4) to give PNA2 (H-g-c-a-a-t-a-a-a-a-M1-lys-NH(2)) and PNA3 (H-P-K-K-K-R-K-V-g-c-a-a-t-a-a-a-a-M1-lys-NH(2)), respectively. The two Ru(II)-PNA oligomers, PNA2 and PNA3, displayed a metal to ligand charge transfer (MLCT) transition band centered around 445 nm and an emission maximum at about 680 nm following 450 nm excitation in aqueous solutions (10 mM PBS, pH 7.4). The absorption and emission response of the duplexes formed with the cDNA strand (DNA: 5'-T-T-T-T-T-T-T-A-T-T-G-C-T-T-T-3') showed no major variations, suggesting that the electronic properties of the Ru(II) complexes are largely unaffected by hybridization. The thermal stability of the PNA·DNA duplexes, as evaluated from UV melting experiments, is enhanced compared to the corresponding nonmetalated duplexes. The melting temperature (T(m)) was almost 8 °C higher for PNA2·DNA duplex, and 4 °C for PNA3·DNA duplex, with the stabilization attributed to the electrostatic interaction between the cationic residues (Ru(II) unit and positively charged lysine/arginine) and the polyanionic DNA backbone. In presence of tripropylamine (TPA) as co-reactant, PNA2, PNA3, PNA2·DNA and PNA3·DNA displayed strong electrochemiluminescence (ECL) signals even at submicromolar concentrations. Importantly, the combination of spectrochemical, thermal and ECL properties possessed by the Ru(II)-PNA sequences offer an elegant approach for the design of highly sensitive multimodal biosensing tools.     

Determining the electrophoretic mobility and translational diffusion coefficients of DNA molecules in free solution

The free solution mobility of DNA molecules of different molecular weights, the sequence dependence of the mobility, and the diffusion coefficients of small single- and double-stranded DNA (ss- and dsDNA) molecules can be measured accurately by capillary zone electrophoresis, using coated capillaries to minimize the electroosmotic flow (EOF) of the solvent. Very small differences in mobility between various analytes can be quantified if a mobility marker is used to correct for small differences in EOF between successive experiments. Using mobility markers, the molecular weight at which the free solution mobility of dsDNA becomes independent of molecular weight is found to be approximately 170 bp in 40 mM Tris-acetate-EDTA buffer. A DNA fragment containing 170 bp has a contour length of approximately 58 nm, close to the persistence length of DNA under these buffer conditions. Hence, the approach of the free solution mobility of DNA to a plateau value may be associated with the transition from a rod-like to a coil-like conformation in solution. Markers have also been used to determine that the free solution mobilities of ss- and dsDNA oligomers are sequence-dependent. Double-stranded 20-bp oligomers containing runs of three or more adenine residues in a row (A-tracts) migrate somewhat more slowly than 20-mers without A-tracts, suggesting that somewhat larger numbers of counterions are condensed in the ion atmospheres of A-tract DNAs, decreasing their net effective charge. Single-stranded 20-mers with symmetric sequences migrate approximately 1% faster than their double-stranded counterparts, and faster than single-stranded 20-mers containing A(5)- or T(5)-tracts. Interestingly, the average mobility of two complementary single-stranded 20-mers is equal to the mobility of the double-stranded oligomer formed upon annealing. Finally, the stopped migration method has been used to measure the diffusion coefficients of single- and double-stranded oligomers. The diffusion coefficients of ssDNA oligomers containing 20 nucleotides are approximately 50% larger than those of double-stranded DNA oligomers of the same size, reflecting the greater flexibility of ssDNA molecules. The methods used to carry out these experiments are also described in detail.     

Michael addition for crosslinking of poly(caprolactone)s

Poly(caprolactone) (PCL) networks have received significant attention in the literature because of many emerging potential applications as biodegradable materials. In this study, the Michael addition reaction was used for the first time to synthesize biodegradable networks using crosslinking of acetoacetate‐functionalized PCL (PCL bisAcAc) oligomers with neopentyl glycol diacrylate. Hydroxyl‐terminated PCL telechelic oligomers with number‐average molecular weights ranging from 1000 to 4000 g/mol were quantitatively functionalized with acetoacetate groups using transacetoacetylation. In addition to difunctional PCL oligomers, hydroxyl‐terminated trifunctional star‐shaped PCL oligomers were functionalized with acetoacetate groups. Derivatization of the terminal hydroxyl groups with acetoacetate groups was confirmed using FTIR spectroscopy, ¹H NMR spectroscopy, mass spectrometry, and base titration of hydroxyl end groups. PCL bisAcAc precursors were reacted with neopentyl glycol diacrylate in the presence of an organic base at room temperature. The crosslinking reactions yielded networks with high gel contents (>85%). The thermomechanical properties of the networks were analyzed to investigate the influence of molecular weight between crosslink points. The glass transition and the extent of crystallinity of the PCL networks were dependent on the molecular weight of the PCL segment. Dynamic mechanical analysis indicated that the plateau modulus of the networks was dependent on the molecular weight of PCL, which was related to the crosslink density of the networks. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 5437–5447, 2009     

Synthesis and evaluation of (1S,2R/1R,2S)-aminocyclohexylglycyl PNAs as conformationally preorganized PNA analogues for DNA/RNA recognition

Conformationally constrained cis-aminocyclohexylglycyl PNAs have been designed on the basis of stereospecific imposition of 1,2-cis-cyclohexyl moieties on the aminoethyl segment of aminoethylglycyl PNA (aegPNA). The introduction of the cis-cyclohexyl ring may allow the restriction of the torsion angle beta in the ethylenediamine segment to 60-70 degrees that is prevalent in PNA(2):DNA and PNA:RNA complexes. The synthesis of the optically pure monomers (10a and 10b) is achieved by stereoselective enzymatic hydrolysis of an intermediate ester 2. The chiral PNA oligomers were synthesized with (1S,2R/1R,2S)-aminocyclohexylglycyl thymine monomers in the center and N-terminus of aegPNA. Differential gel shift retardation with one or more units of modified monomer units was observed as a result of hybridization of PNA sequences with complementary DNA sequences. Hybridization studies with complementary DNA and RNA sequences using UV-T(m) measurements indicate that PNA with (1S,2R)-cyclohexyl stereochemistry enhances selective binding with RNA over DNA as compared to control aegPNA and PNA with the other (1R,2S) isomer.     

Monitoring oligomer formation from self-aggregating amylin peptides using ESI-IMS-MS

Amyloid diseases are a serious cause for concern world-wide. To understand the mechanism of formation of the fibrillar structures associated with such disorders, it is necessary to study the progression from soluble protein or peptide monomer through an array of oligomers to the final, insoluble, fibrils. The protein IAPP is found in vivo in the form of insoluble amyloid deposits in the pancreatic islets of diabetes type II sufferers. Here, we have studied the in vitro self-aggregation of three fibril-forming peptides from the amyloidogenic core of IAPP. Using electrospray ionization—mass spectrometry coupled with ion mobility spectrometry, the mass and cross-sectional area of each oligomer present in the heterogeneous assembly mixtures can be determined individually in a single, rapid experiment over time. For the three peptides studied, oligomers ≤20-mer were characterized. Conversely, no oligomers higher than a dimer were detected for a non-assembling peptide control. The rate in which the cross-sectional area of the oligomers increases with increasing number of peptide sub-units indicates that assembly for the amyloid-forming peptides proceeds in a linear fashion until an oligomer of a certain size is attained. After this, a step increase in cross-sectional area occurs for the next higher-order oligomer. This behaviour can be explained by molecular modelling of singly, doubly, triply and quadruply stacked β-stranded structures. Using one peptide as an example, the cross-sectional areas of the lower order oligomers (dimer to pentamer) were found to be consistent with a single β-sheet model, whereas the higher order oligomers were consistent with double-stranded (hexamer to decamer oligomers), triply-stranded (11-mers to 15-mers) and quadruply-stranded (16-mers to 20-mers) β-sheet models.     

Guanosine gel for sequence-dependent separation of polymorphic ssDNA

The separation of fluorescent-labeled ssDNA fragments of equal length based on differences in sequence was achieved through the use of guanosine gels (G-gels) formed by guanosine-5'-monophosphate (GMP) in capillary gel electrokinetic chromatography (CGEKC) with LIF detection. Baseline resolution was achieved for homodimers and homopentamers of A, T, and C. G-gel CGEKC provided better resolution than CZE, MEKC, or a sieving gel in CGE. Resolution improved with increasing GMP, indicating that the interaction is linked to structural organization of the G-gel. Fluorescence intensity and anisotropy show that the order of interaction with G-gels is T>C>A. We then investigated four conformationally similar, polymorphic 76-mers with A/G substitutions that are utilized in forensic DNA typing. Resolution was achieved by CGEKC but not CZE or CGE. In CGEKC, the negatively charged G-gels and oligonucleotides electromigrate toward the positive inlet while being driven by EOF to the negative outlet. The net forward velocity is the greatest for oligonucleotides most closely associated with the slower, more cumbersome G-gel network. For the 76-mers, resolution increases with increasing difference in guanosine content between strands and, for a given difference, with increasing total guanosines in the strands.     

MALDI analysis of oligonucleotides directly from montmorillonite

Oligonucleotides synthesized on a montmorillonite catalyst were analyzed directly. By mixing the catalyst with a matrix (2,4,6-trihydroxyacetophenone or 6-aza-2-thiothymine) and dibasic ammonium citrate, higher molecular weight products were detected compared with "classical" methods such as gel electrophoresis and HPLC with UV as a detector. The oligomers (30-mers and higher) were detected by mass spectrometry even though their concentration was less than 10(-4)% of the total content of the RNA. This method is different from the (MALDI) analysis of the eluates from montmorillonite, which otherwise requires desalting. Placing reaction mixtures with a high concentration of buffers on homoionic, preferably Li-containing, montmorillonite does not require desalting.     

Convergent strategies for the attachment of fluorescing reporter groups to peptide nucleic acids in solution and on solid phase

The site-selective conjugation of peptide nucleic acids (PNA) with fluorescent reporter groups is essential for the construction of hybridisation probes that can report the presence of a particular DNA sequence. This paper describes convergent methods for the solution- and solid-phase synthesis of multiply labelled PNA oligomers. The solid-phase synthesis of protected PNA enabled the selective attachment of fluorescent labels at the C-terminal end (3' in DNA) which demonstrated that further manipulations on protected PNA fragments are feasible. For the conjugation to internal sites, a method is introduced that allows for the on-resin assembly of modified monomers thereby omitting the need to synthesise an entire monomer in solution. Furthermore, it is shown that the application of a highly orthogonal protecting group strategy in combination with chemoselective conjugation reactions provides access to a rapid and automatable solid-phase synthesis of dual labelled PNA probes. Real-time measurements of nucleic acid hybridisation were possible by taking advantage of the fluorescence resonance energy transfer (FRET) between suitably appended fluorophoric groups. Analogously to DNA-based molecular beacons, the dual labelled PNA probes were only weakly fluorescing in the single-stranded state. Hybridisation to a complementary oligonucleotide, however, induced a structural reorganisation and conferred a vivid fluorescence enhancement.