Two-dimensional LNA/DNA arrays: estimating the helicity of LNA/DNA hybrid duplex

We measured the helical repeats of a non-natural nucleic acid, locked nucleic acid (LNA), by incorporating LNA strands into the outer arms of a DNA double crossover (DX) molecule; atomic force microscopy (AFM) imaging of the two-dimensional (2D) arrays self-assembled from these DX molecules allows us to derive the helical repeat of the LNA/DNA hetero-duplex to be 13.2 +/- 0.9 base pairs per turn.     

Pyrrolidine nucleic acids: DNA/PNA oligomers with 2-hydroxy/aminomethyl- 4-(thymin-1-yl)pyrrolidine-N-acetic acid

Synthesis of pyrrolidine-based chiral positively charged DNA analogues is reported. The synthesis of (2S,4S) and (2R,4R) thymin-1-ylpyrrolidine-N-acetic acid, its site specific incorporation in PNA:DNA chimera and PNA, and the study of their binding properties with complementary DNA/RNA sequences is presented.     

PNA encoding (PNA=peptide nucleic acid): from solution-based libraries to organized microarrays

Microarray-based technologies have attracted attention in chemical biology by virtue of their miniaturized format, which is well suited to probe ligand-protein interactions or investigate enzymatic activity in complex biological mixtures. A number of research groups have reported the preparation of surfaces on microarrays with specific functional groups to chemoselectively attach small molecules from libraries. We have developed an alternative method whereby libraries are encoded with peptide nucleic acid (PNA), such that libraries which exist as mixtures in solution self-assemble into an organized microarray through hybridization to produce readily available DNA arrays. This allows libraries synthesized by split and mix methods to be decoded in a single step. An asset of this method compared to direct spotting is that libraries can be used in solution for bioassays prior to self-assembly into the microarray format.     

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.     

Charge dependent behavior of PNA/DNA/PNA triplexes in the gas phase

Intact noncovalent complexes were studied in the gas phase using negative ion nano-ESI mass spectrometry. Among various noncovalent systems studied in the gas phase, the interaction of DNA strands with peptide nucleic acids (PNAs) presents a strong interest as biologically relevant systems. PNAs originally described by Nielsen are used as DNA mimics as possible medical agents by imprisoning DNA single strands into stable noncovalent complexes. Two types of PNAs were investigated in the PNA/DNA multiplex: the original Nielsen's PNA and a modified backbone PNA by the introduction of syn- and anti-(aminoethyl)thiazolidine rings. We first investigated the stoichiometry of PNA/DNA multiplexes formed in solution and observed them in the gas phase via qualitative kinetics of complementary strand associations. It resulted in observing PNA2/DNA triplexes (ts) as the multiply deprotonated species, most stable in both the solution and gas phase. Second, charge-dependant decompositions of these species were undertaken under low-energy collision conditions. It appears that covalent bond cleavages (base releasing or skeleton cleavage) occur from lower ts charge states rather than ts unzipping, which takes place from higher charge states. This behavior can be explained by considering the presence of zwitterions depending on the charge state. They result in strong salt-bridge interactions between the positively charged PNA side chain and the negatively charged DNA backbone. We propose a general model to clearly display the involved patterns in the noncovalent triplex decompositions. Third, the relative stability of three PNA2/DNA complexes was scrutinized in the gas phase by acquiring the breakdown curves of their ts(6-) form, corresponding to the ts unzipping. The chemical structures of the studied PNAs were chosen in order to evidence the possible influence of backbone stereochemistry on the rigidity of PNA2/DNA complexes. It provided significantly different stabilities via V(m) measurements. The relative gas-phase stability order obtained was compared to that found in solution by Chassaing et al., and shows qualitative agreement.     

Synthesis and nucleic acid binding studies of novel pyrrolidinyl PNA carrying an N-amino-N-methylglycine spacer

Two novel pyrrolidinyl peptide nucleic acids comprising alternating sequences of thymine-modified d- or l-proline and an N-amino-N-methylglycine spacer were synthesized using solid-phase methodology. UV and CD titrations together with a gel-binding shift assay revealed that neither of the homothymine PNA decamers bind to their complementary DNA or RNA. This was considered to be due to an unfavorable secondary structure which could not be alleviated by the presence of the positively charged protonated amine in the PNA backbone.     

Efficient functional molecule incorporation method to functionalized peptide nucleic acid (PNA): use in synthesis of labeled PNA oligomers

A novel efficient synthetic method for a functionalized PNA (peptide nucleic acid) is described, in which a functional molecule is incorporated in place of a nucleobase. Novel ω-AA-^BocPNA-OH (20-24, AA=amino acid) were designed as PNA precursor monomer units into which functional molecules could be incorporated efficiently. Compounds 20-24 reacted quantitatively with OSu (N-hydroxysuccinimidyl) active ester derivatives and isothiocyanate derivatives of commercial functional molecules to give target functionalized PNA monomer units 25-53. Various types of functionalized PNA monomer units could be efficiently incorporated into multiple predetermined positions in a PNA oligomer by SPPS (solid phase peptide synthesis) in the same way as for the four A(Cbz), G(Cbz), C(Cbz), and T PNA monomer units.     

Direction control in DNA binding of chiral D-lysine-based peptide nucleic acid (PNA) probed by electrospray mass spectrometry

The DNA binding abilities of peptide nucleic acids (PNAs), both achiral and bearing three adjacent D-lysine-based monomers in the middle of the strand ("chiral box" PNA), were studied by means of electrospray mass spectrometry (ESI-MS). In contrast with achiral PNA, "Chiral box" PNA was confirmed to exert high direction control (antiparallel vs. parallel DNA target) in DNA binding.     

Graft copolymers of nucleic acid bases on polyethyleneimine: Interaction of the polymers

Interactions of the graft copolymers of nucleic acid bases on polyethyleneimine with polynucleotides were studied. The backbone of the polymer is linear or branched polyethyleneimine. The nucleic acid bases are adenine and thymine. The polynucleotides used were polyadenylic acid (poly-A) and polyuridylic acid (poly-U). Interactions between these polymers were measured by ultraviolet (UV) absorption and the results were compared with the vinyl-type nucleic acid model polymer.     

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.     

Expanding the repertoire of pyrrolidyl PNA analogues for DNA/RNA hybridization selectivity: aminoethylpyrrolidinone PNA (aepone-PNA)

New PNA analogues derived from aminoethylpyrrolidin-5-one backbone show stabilization of aepone-PNA:DNA hybrids and destabilization of the corresponding RNA hybrids compared to unmodified PNA.     

Synthesis of hydrazino-peptide nucleic acid monomers and dimers as new PNA backbone building blocks

We describe the synthesis of new hydrazinoPNA (hydPNA) monomers and new hydPNA-containing dimers. For the hydPNA monomers, the primary terminal amino group of the aminoethylglycine unit of classical aegPNA is replaced by a hydrazine moiety. An appropriate choice of two orthogonal protecting groups on the two hydrazine nitrogen atoms makes it possible to drive their coupling with other monomers selectively on one or the other nitrogen atom, thus obtaining two different types of PNA dimers. These dimers represent new building blocks that can be used to generate novel PNA oligomers.     

cis-Cyclopentyl PNA (cpPNA) as constrained chiral PNA analogues: stereochemical dependence of DNA/RNA hybridization

DNA/RNA hybridization studies of PNA-T oligomers with cis-(1S,2R/1R,2S)-cyclopentyl units in the backbone show stereochemistry dependent binding with RNA/DNA discrimination.     

Gene sensors based on peptide nucleic acid (PNA) probes: relationship between sensor sensitivity and probe/target duplex stability

Gene sensors based on peptide nucleic acid (PNA) probes were prepared and the relationship between sensor sensitivity and the duplex stability of the probe PNAs and target complementary DNAs was studied using five synthesized PNAs (10-, 15-, 17-, 20-, and 22-mers). It was found that the association constants for the probe PNA/target DNA pairs depend not only on the length but also on the base pair sequence, and that the trend in the sensor responses was the same as that in the association constants for the corresponding pairs. In addition, by using two kinds of probe PNAs with different lengths, it was demonstrated that fabrication of sensors based on probe PNAs with comparable association constants yielded similar response curves and sensor sensitivities.     

Cyanuryl peptide nucleic acid: synthesis and DNA complexation properties

The synthesis of cyanuryl PNA monomer (CyaPNA) 6 was achieved by direct N-monoalkylation of cyanuric acid with N-(2-Boc-aminoethyl)-N′-(bromoacetyl)glycyl ethyl ester 4. Compound 6 was incorporated as a T-mimic into PNA oligomers and biophysical studies on their triplexes/duplex complexes with complementary DNA oligomers indicated unusual stabilization of PNA:DNA hybrids when the cyanuryl unit was located in the middle of the PNA oligomer.     

DNA nanostructure-based nucleic acid probes: construction and biological applications

In recent years, DNA has been widely noted as a kind of material that can be used to construct building blocks for biosensing, in vivo imaging, drug development, and disease therapy because of its advantages of good biocompatibility and programmable properties. However, traditional DNA-based sensing processes are mostly achieved by random diffusion of free DNA probes, which were restricted by limited dynamics and relatively low efficiency. Moreover, in the application of biosystems, single-stranded DNA probes face challenges such as being difficult to internalize into cells and being easily decomposed in the cellular microenvironment. To overcome the above limitations, DNA nanostructure-based probes have attracted intense attention. This kind of probe showed a series of advantages compared to the conventional ones, including increased biostability, enhanced cell internalization efficiency, accelerated reaction rate, and amplified signal output, and thus improved in vitro and in vivo applications. Therefore, reviewing and summarizing the important roles of DNA nanostructures in improving biosensor design is very necessary for the development of DNA nanotechnology and its applications in biology and pharmacology. In this perspective, DNA nanostructure-based probes are reviewed and summarized from several aspects: probe classification according to the dimensions of DNA nanostructures (one, two, and three-dimensional nanostructures), the common connection modes between nucleic acid probes and DNA nanostructures, and the most important advantages of DNA self-assembled nanostructures in the applications of biosensing, imaging analysis, cell assembly, cell capture, and theranostics. Finally, the challenges and prospects for the future development of DNA nanostructure-based nucleic acid probes are also discussed.

In recent years, DNA has been widely noted as a kind of material that can be used to construct building blocks for biosensing, in vivo imaging, drug development, and disease therapy because of its advantages of good biocompatibility and programmable properties.     

Aminoethylprolyl peptide nucleic acids (aepPNA): chiral PNA analogues that form highly stable DNA:aepPNA2 triplexes

[formula: see text] The replacement of the glycyl component in the peptide nucleic acid (PNA) backbone by a prolyl unit bearing a nucleobase leads to the aminoethylprolyl (aep) PNAs, which are chiral and cationic. The homooligomeric aepPNA binds to complementary DNA sequences with high affinity and sequence specificity, forming highly stable triplexes.     

Aminoethylprolyl (aep) PNA: mixed purine/pyrimidine oligomers and binding orientation preferences for PNA:DNA duplex formation

[structure in text] The synthesis of (2S,4S)- and (2R,4S)-aepPNA monomers of adenine, guanine, and cytosine (3-5) and their incorporation at appropriate positions into aegPNA sequence 7 leads to mixed aeg-aep backbone/mixed nucleobase PNAs 8-11. The thermal stabilities of the derived duplexes with DNA are found to be dependent on nucleobase and backbone stereochemistry.