Multilabeled pyrene-functionalized 2'-amino-LNA probes for nucleic acid detection in homogeneous fluorescence assays

Homogeneous fluorescence assays for detection of nucleic acids are widely used in biological sciences. Typically, probes such as molecular beacons that rely on distance-dependent fluorescence quenching are used for such assays. Less attention has been devoted to tethering a single kind of fluorophores to oligonucleotides and exploiting hybridization-induced modulation of fluorescence intensity for nucleic acid detection. Herein, thermal denaturation experiments and fluorescence properties of oligodeoxyribonucleotides containing one or more 2'-N-(pyren-1-yl)carbonyl-2'-amino-LNA monomer(s) X are described. These pyrene-functionalized 2'-amino-LNAs display large increases in thermal stability against DNA/RNA complements with excellent Watson-Crick mismatch discrimination. Upon duplex formation of appropriately designed 2'-N-(pyren-1-yl)carbonyl-2'-amino-LNA probes and complementary DNA/RNA, intensive fluorescence emission with quantum yields between 0.28 and 0.99 are observed. Quantum yields of such magnitudes are unprecedented among pyrene-labeled oligonucleotides. Molecular modeling studies suggest that the dioxabicyclo[2.2.1]heptane skeleton and amide linkage of monomer X fix the orientation of the pyrene moiety in the minor groove of a nucleic acid duplex. Interactions between pyrene and nucleobases, which typically lead to quenching of fluorescence, are thereby reduced. Duplexes between multiple modified probes and DNA/RNA complements exhibit additive increases in fluorescence intensity, while the fluorescence of single stranded probes becomes increasingly quenched. Up to 69-fold increase in fluorescence intensity (measured at lambda(em) = 383 nm) is observed upon hybridization to DNA/RNA. The emission from duplexes of multiple modified probes and DNA/RNA at concentrations down to less than 500 nM can easily be seen by the naked eye using standard illumination intensities.     

Pyrene excimer signaling molecular beacons for probing nucleic acids

Molecular beacon DNA probes, containing 1-4 pyrene monomers on the 5' end and the quencher DABCYL on the 3' end, were engineered and employed for real-time probing of DNA sequences. In the absence of a target sequence, the multiple-pyrene labeled molecular beacons (MBs) assumed a stem-closed conformation resulting in quenching of the pyrene excimer fluorescence. In the presence of target, the beacons switched to a stem-open conformation, which separated the pyrene label from the quencher molecule and generated an excimer emission signal proportional to the target concentration. Steady-state fluorescence assays resulted in a subnanomolar limit of detection in buffer, whereas time-resolved signaling enabled low-nanomolar target detection in cell-growth media. It was found that the excimer emission intensity could be scaled by increasing the number of pyrene monomers conjugated to the 5' terminal. Each additional pyrene monomer resulted in substantial increases in the excimer emission intensities, quantum yields, and excited-state lifetimes of the hybridized MBs. The long fluorescence lifetime ( approximately 40 ns), large Stokes shift (130 nm), and tunable intensity of the excimer make this multiple-pyrene moiety a useful alternative to traditional fluorophore labeling in nucleic acid probes.     

C5-functionalized DNA, LNA, and α-L-LNA: positional control of polarity-sensitive fluorophores leads to improved SNP-typing

Single nucleotide polymorphisms (SNPs) are important markers in disease genetics and pharmacogenomic studies. Oligodeoxyribonucleotides (ONs) modified with 5-[3-(1-pyrenecarboxamido)propynyl]-2'-deoxyuridine monomer X enable detection of SNPs at non-stringent conditions due to differential fluorescence emission of matched versus mismatched nucleic acid duplexes. Herein, the thermal denaturation and optical spectroscopic characteristics of monomer X are compared to the corresponding locked nucleic acid (LNA) and α-L-LNA monomers Y and Z. ONs modified with monomers Y or Z result in a) larger increases in fluorescence intensity upon hybridization to complementary DNA, b) formation of more brightly fluorescent duplexes due to markedly larger fluorescence emission quantum yields (Φ(F)=0.44-0.80) and pyrene extinction coefficients, and c) improved optical discrimination of SNPs in DNA targets. Optical spectroscopy studies suggest that the nucleobase moieties of monomers X-Z adopt anti and syn conformations upon hybridization with matched and mismatched targets, respectively. The polarity-sensitive 1-pyrenecarboxamido fluorophore is, thereby, either positioned in the polar major groove or in the hydrophobic duplex core close to quenching nucleobases. Calculations suggest that the bicyclic skeletons of LNA and α-L-LNA monomers Y and Z influence the glycosidic torsional angle profile leading to altered positional control and photophysical properties of the C5-fluorophore.     

Modulation of pyrene fluorescence in DNA probes depends upon the nature of the conformationally restricted nucleotide

The DNA probes (ODNs) containing a 2'-N-(pyren-1-yl)-group on the conformationally locked nucleosides [2'-N-(pyren-1-yl)carbonyl-azetidine thymidine, Aze-pyr (X), and 2'-N-(pyren-1-yl)carbonyl-aza-ENA thymidine, Aza-ENA-pyr (Y)], show that they can bind to complementary RNA more strongly than to the DNA. The Aze-pyr (X) containing ODNs with the complementary DNA and RNA duplexes showed an increase in the fluorescence intensity (measured at lambda em approximately 376 nm) depending upon the nearest neighbor at the 3'-end to X [dA ( approximately 12-20-fold) > dG ( approximately 9-20-fold) > dT ( approximately 2.5-20-fold) > dC ( approximately 6-13-fold)]. They give high fluorescence quantum yields (Phi F = 0.13-0.89) as compared to those of the single-stranded ODNs. The Aza-ENA-pyr (Y)-modified ODNs, on the other hand, showed an enhancement of the fluorescence intensity only with the complementary DNA (1.4-3.9-fold, Phi F = 0.16-0.47); a very small increase in fluorescence is also observed with the complementary RNA (1.1-1.7-fold, Phi F = 0.17-0.22), depending both upon the site of the Y modification introduced as well as on the chemical nature of the nucleobase adjacent to the modification site into the ODN. The fluorescence properties, thermal denaturation experiments, absorption, and circular dichroism (CD) studies with the X- and Y-modified ODNs in the form of matched homo- and heteroduplexes consistently suggested (i) that the orientation of the pyrene moiety is outside the helix of the nucleic acid duplexes containing a dT-d/rA base pair at the 3'-end of the modification site for both X and Y types of modifications, and (ii) that the microenvironment around the pyrene moiety in the ODN/DNA and ODN/RNA duplexes is dictated by the chemical nature of the conformational constraint in the sugar moiety, as well as by the nature of neighboring nucleobases. The pyrene fluorescence emission in both X and Y types of the conformationally restricted nucleotides is found to be sensitive to a mismatched base present in the target RNA: (i) The X-modified ODN showed a decrease ( approximately 37-fold) in the fluorescence intensity (measured at lambda em approximately 376 nm) upon duplex formation with RNA containing a G nucleobase mismatch (dT-rG pair instead of dT-rA) opposite to the modification site. (ii) In contrast, the Y-modified ODN in the heteroduplex resulted in a approximately 3-fold increase in the fluorescence intensity upon dT-rG mismatch, instead of matched dT-rA pair, in the RNA strand. Our data corroborate that the pyrene moiety is intercalated in the X-modified mismatched ODN/RNA (G mismatch) heteroduplex as compared to that of the Y-modified ODN/RNA (G mismatch) heteroduplex, in which it is located outside the helix.     

Novel interstrand communication systems within DNA duplexes based on 1-, 2- and 4-(phenylethynyl)pyrenes attached to 2'-amino-LNA: high-affinity hybridization and fluorescence sensing

Functionalisation of 2'-amino-LNA oligonucleotides with 1-, 2- and 4-(phenylethynyl)pyrene fluorophores via a carbonyl linker (PEPyc) resulted in efficient interstrand communication systems in nucleic acid duplexes, providing effective tools for stabilization of nanostructures and fluorescence monitoring of DNA self-assembly.     

Dual pyrene-labeled pyrrolidinyl peptide nucleic acid as an excimer-to-monomer switching probe for DNA sequence detection

The unique ability of pyrene to form excimers with distinct emission characteristic from monomer offers an attractive means to signal the interactions between biomolecules. In this work, dual pyrene-labeled pyrrolidinyl peptide nucleic acid probe with a d-prolyl-2-aminocyclopetanecarboxylic acid α,β-dipeptide backbone (acpcPNA) was designed as an excimer-to-monomer switching probe for DNA sequence detection. In single stranded state, the excimer emission at 470 nm was mainly observed in the fluorescence spectrum. In the presence of DNA target, the hybridization resulted in separation of the two pyrene units, therefore the spectrum displayed increased monomer emission at 380 nm with concomitant decreased excimer emission. Switching ratio, which is defined as the ratio of the monomer to excimer in the double stranded form [F₃₈₀/F₄₇₀(ds)] divided by the same value obtained from the single stranded form [F₃₈₀/F₄₇₀(ss)], was used to describe the performance of the probes. Switching ratios in the range of 5–30 were observed with various dual pyrene-labeled acpcPNA probes bearing pyrenebutyryl label attached five-base apart. Practically no excimer-to-monomer switching behavior was observed with DNA targets carrying a single mismatched base as shown by the small switching ratios of ∼1.     

Synthesis and RNA-selective hybridization of α-l-ribo- and β-d-lyxo-configured oligonucleotides

Three α-l-ribofuranosyl analogues of RNA nucleotides (α-l-RNA analogues) have been synthesized and incorporated into oligonucleotides using the phosphoramide approach on an automated DNA synthesizer. The 4′-C-hydroxymethyl-α-l-ribofuranosyl thymine monomer was furthermore synthesized. Relative to the unmodified duplexes, incorporation of a single α-l-RNA monomer into a DNA strand leads to reduced thermal stability of duplexes with DNA complements but unchanged thermal stability of duplexes with RNA complements, whereas incorporation of more than one α-l-RNA monomer lead to moderately decreased thermal stability also of duplexes with RNA complements. Efficient hybridization with an RNA complement and no melting transition with a DNA complement were observed with stereoregular chimeric oligonucleotides composed of a mixture of α-l-RNA and affinity enhancing α-l-LNA monomers (α-l-ribo-configured locked nucleic acid). Furthermore, duplexes formed between oligodeoxynucleotides containing an α-l-RNA monomer and complementary RNA were good substrates for Escherichia coli RNase H. RNA-selective hybridization was also achieved by the incorporation of 1-(4-C-hydroxymethyl-β-d-lyxofuranosyl)thymine monomers into a DNA strand, whereas stable duplexes were formed with both complementary DNA and RNA when these monomers were incorporated into an RNA strand.     

Unlocked nucleic acid--an RNA modification with broad potential

The first unlocked nucleic acid (UNA) monomer was described more than a decade ago, but only recent reports have revealed the true potential applications of this acyclic RNA mimic. UNA monomers enable the modulation of the thermodynamic stability of various nucleic acid structures such as RNA and DNA duplexes, quadruplexes or i-motifs. Moreover, UNA monomers were found to be compatible with RNase H activity, a property which is important for single stranded antisense constructs. Notably, UNA monomers can be applied in the design of superior siRNAs, combining potent gene silencing and dramatically reduced off-target effects.     

On the stability of double stranded nucleic acids

We present the first pressure-versus-temperature phase diagram for the helix-to-coil transition of double stranded nucleic acids. The thermodynamic stability of a nucleic acid duplex is a complex function of temperature and pressure and strongly depends on the denaturation temperature, T(M), of the duplex at atmospheric pressure. Depending upon T(M), pressure, and temperature, the phase diagram shows that pressure may stabilize, destabilize, or have no effect on the conformational state of DNA. To verify the phase diagram, we have conducted high-pressure UV melting experiments on poly(dIdC)poly(dIdC), a DNA duplex, poly(rA)poly(rU), an RNA duplex, and poly(dA)poly(rU), a DNA/RNA hybrid duplex. The T(M) values of these duplexes have been modulated by altering the solution ionic strength. Significantly, at low salt, these three duplexes have helix-to-coil transition temperatures of 50 degrees C or less. In agreement with the derived phase diagram, we found that the polymeric duplexes were destabilized by pressure if the T(M) is < approximately 50 degrees C. However, these duplexes were stabilized by pressure if the T(M) is > approximately 50 degrees C. The DNA/RNA hybrid duplex, poly(dA)poly(rU), with a T(M) of 31 degrees C in 20 mM NaCl undergoes a pressure-induced helix-to-coil transition at room temperature. This is the first report of pressure-induced denaturation of a nucleic acid duplex and provides new insights into the molecular forces stabilizing these structures.     

C2'-pyrene-functionalized triazole-linked DNA: universal DNA/RNA hybridization probes

Development of universal hybridization probes, that is, oligonucleotides displaying identical affinity toward matched and mismatched DNA/RNA targets, has been a longstanding goal due to potential applications as degenerate PCR primers and microarray probes. The classic approach toward this end has been the use of "universal bases" that either are based on hydrogen-bonding purine derivatives or aromatic base analogues without hydrogen-bonding capabilities. However, development of probes that result in truly universal hybridization without compromising duplex thermostability has proven challenging. Here we have used the "click reaction" to synthesize four C2'-pyrene-functionalized triazole-linked 2'-deoxyuridine phosphoramidites. We demonstrate that oligodeoxyribonucleotides modified with the corresponding monomers display (a) minimally decreased thermal affinity toward DNA/RNA complements relative to reference strands, (b) highly robust universal hybridization characteristics (average differences in thermal denaturation temperatures of matched vs mismatched duplexes involving monomer W are <1.7 °C), and (c) exceptional affinity toward DNA targets containing abasic sites opposite of the modification site (ΔT(m) up to +25 °C). The latter observation, along with results from absorption and fluorescence spectroscopy, suggests that the pyrene moiety is intercalating into the duplex whereby the opposing nucleotide is pushed into an extrahelical position. These properties render C2'-pyrene-functionalized triazole-linked DNA as promising universal hybridization probes for applications in nucleic acid chemistry and biotechnology.     

Synthesis and properties of trans-3',4'-bridged nucleic acids having typical S-type sugar conformation

The synthesis of nucleoside analogues with a conformationally restricted sugar moiety is of great interest. The present research describes the synthesis of BNA (bridged nucleic acid) monomers 1 and 2 bearing a 4,7-dioxabicyclo[4.3.0]nonane skeleton and a methoxy group at the C2' position. Conformational analysis showed that the sugar moiety of these monomers is restricted in a typical S-type conformation. It was difficult to synthesize the phosphoramidite derivative of the ribo-type monomer 1, while the phosphoramidite of the arabino-type monomer 2 was successfully prepared and incorporated into oligodeoxynucleotides (ODNs). The hybridization ability of the obtained ODN derivatives containing 2 with complementary strands was evaluated by melting temperature (T(m)) measurements. As a result, the ODN derivatives hybridized with DNA and RNA complements in a sequence-selective manner, though the stability of the duplexes was lower than that of the corresponding natural DNA/DNA or DNA/RNA duplex.     

Interstrand communication between 2'-N-(pyren-1-yl)methyl-2'-amino-LNA monomers in nucleic acid duplexes: directional control and signalling of full complementarity

Very efficient interstrand communication systems in nucleic acid duplexes, based on pyrene excimer formation between 2'-N-(pyren-1-yl)methyl-2'-amino-LNA monomers, demonstrate the versatility of functionalized 2'-amino-LNA monomers for Angstrom-scale chemical engineering.     

Analogues of a locked nucleic acid with three-carbon 2',4'-linkages: synthesis by ring-closing metathesis and influence on nucleic acid duplex stability and structure

Two bicyclic 2'-deoxynucleoside analogues containing a saturated and an unsaturated three-carbon 2',4'-linkage, respectively, have been synthesized using a ring-closing metathesis-based linear strategy starting from uridine. Both analogues have been incorporated into oligodeoxynucleotide sequences and increased the stability of DNA:RNA hybrid duplexes (DeltaT(m) approximately 2.5-5.0 degrees C per modification) and decreased the stability of dsDNA duplexes (DeltaT(m) approximately 2.5-1.0 degrees C per modification). CD spectroscopy revealed that the bicyclic nucleosides induced formation of A-type-like duplexes albeit to a lesser degree than found for locked nucleic acid (LNA) monomers. From the CD data and UV melting analysis, we propose that the 2'-oxygen atom of the bicyclic moiety is essential for the formation of stabilized A-type-like dsDNA but not for the formation of a stabilized A-type DNA:RNA hybrid.     

Synthesis and oligomerization of Fmoc/Boc-protected PNA monomers of 2,6-diaminopurine, 2-aminopurine and thymine

A Boc-protecting group strategy for Fmoc-based PNA (peptide nucleic acid) oligomerization has been developed for thymine, 2,6-diaminopurine (DAP) and 2-aminopurine (2AP). The monomers may be used interchangeably with standard Fmoc PNA monomers. The DAP monomer was incorporated into a PNA and was found to selectively bind to T (ΔT(m)≥ +6 °C) in a complementary DNA strand. The 2AP monomer showed excellent discrimination of T (ΔT(m)≥ +12 °C) over the other nucleobases. 2AP also acted as a fluorescent probe of the PNA:DNA duplexes and displayed fluorescence quenching dependent on the opposite base.     

Nonenzymatic synthesis of RNA and DNA oligomers on hexitol nucleic acid templates: the importance of the A structure.

Hexitol nucleic acid (HNA) is an analogue of DNA containing the standard nucleoside bases, but with a phosphorylated 1,5-anhydrohexitol backbone. HNA oligomers form duplexes having the nucleic acid A structure with complementary DNA or RNA oligomers. The HNA decacytidylate oligomer is an efficient template for the oligomerization of the 5'-phosphoroimidazolides of guanosine or deoxyguanosine. Comparison of the oligomerization efficiencies on HNA, RNA, and DNA decacytidylate templates under various conditions suggests strongly that only nucleic acid double helices with the A structure support efficient template-directed synthesis when 5'-phosphoroimidazolides of nucleosides are used as substrates.     

Synthesis and photo activity of phenylazonaphthalene peptide nucleic acid monomers

Phenylazonaphthalene peptide nucleic acid （PNA） monomers were successfully synthesized, and their photoisomerization was examined. The new PNA monomers showed reversible trans-cis isomerization with UVand visible light irradiation, which might be the foundation of photo-regulating the hybridization between PNA containing phenylazonaphthalene unit and DNA. Simultaneously, the fluorescence of the new PNA monomers might make them especially useful as structural probes.     

Incorporation of Pseudo‐complementary Bases 2,6‐Diaminopurine and 2‐Thiouracil into Serinol Nucleic Acid (SNA) to Promote SNA/RNA Hybridization

Serinol nucleic acid (SNA) is a promising candidate for nucleic acid‐based molecular probes and drugs due to its high affinity for RNA. Our previous work revealed that incorporation of 2,6‐diaminpurine (D), which can form three hydrogen bonds with uracil, into SNA increases the melting temperature of SNA‐RNA duplexes. However, D incorporation into short self‐complementary regions of SNA promoted self‐dimerization and hindered hybridization with RNA. Here we synthesized a SNA monomer of 2‐thiouracil (sU), which was expected to inhibit base pairing with D by steric hindrance between sulfur and the amino group. To prepare the SNA containing D and sU in high yield, we customized the protecting groups on D and sU monomers that can be readily deprotected under acidic conditions. Incorporation of D and sU into SNA facilitated stable duplex formation with target RNA by suppressing the self‐hybridization of SNA and increasing the stability of the heteroduplex of SNA and its complementary RNA. Our results have important implications for the development of SNA‐based probes and nucleic acid drugs.     

Metal-containing peptide nucleic acid conjugates

Peptide Nucleic Acids (PNAs) are non-natural DNA/RNA analogues with favourable physico-chemical properties and promising applications. Discovered nearly 20 years ago, PNAs have recently re-gained quite a lot of attention. In this Perspective article, we discuss the latest advances on the preparation and utilisation of PNA monomers and oligomers containing metal complexes. These metal- conjugates have found applications in various research fields such as in the sequence-specific detection of nucleic acids, in the hydrolysis of nucleic acids and peptides, as radioactive probes or as modulators of PNA·DNA hybrid stability, and last but not least as probes for molecular and cell biology.     

alpha-L-ribo-configured locked nucleic acid (alpha-L-LNA): synthesis and properties

The syntheses of monomeric nucleosides and 3'-O-phosphoramidite building blocks en route to alpha-L-ribo-configured locked nucleic acids (alpha-L-LNA), composed entirely of alpha-L-LNA monomers (alpha-L-ribo configuration) or of a mixture of alpha-L-LNA and DNA monomers (beta-D-ribo configuration), are described and the alpha-L-LNA oligomers are studied. Bicyclic 5-methylcytosin-1-yl and adenine-9-yl nucleoside derivatives have been prepared and the phosphoramidite approach has been used for the automated oligomerization leading to alpha-L-LNA oligomers. Binding studies revealed very efficient recognition of single-stranded DNA and RNA target oligonucleotide strands. Thus, stereoirregular alpha-L-LNA 11-mers containing a mixture of alpha-L-LNA monomers and DNA monomers ("mix-mer alpha-L-LNA") were shown to display DeltaT(m) values of +1 to +3 degrees C per modification toward DNA and +4 to +5 degrees C toward RNA when compared with the corresponding unmodified DNA x DNA and DNA x RNA reference duplexes. The corresponding DeltaT(m) values per modification for the stereoregular fully modified alpha-L-LNA were determined to be +4 degrees C (against DNA) and +5 degrees C (against RNA). 11-Mer alpha-L-LNAs (mix-mer alpha- L-LNA or fully modified alpha- L-LNA) were shown in vitro to be significantly stabilized toward 3'-exonucleolytic degradation. A duplex formed between RNA and either mix-mer alpha-L-LNA or fully modified alpha-L-LNA induced in vitro Escherichia coli RNase H-mediated cleavage, albeit very slow, of the RNA targets at high enzyme concentrations.     

Polyaniline based nucleic acid sensor

Twenty-bases long NH2-modified DNA and PNA probes specific to a pathogen (Mycobacterium tuberculosis) were covalently immobilized onto a polyaniline (PANI)/Au electrode to detect nucleic acid hybridization with complementary, one-base mismatch and noncomplementary targets within 30 s using Methylene Blue. The PNA-PANI/Au electrode exhibits improved specificity (1000 times) and detection limit (0.125 x 10(-18) M) as compared to that of the DNA-PANI/Au electrode (2.5 x 10(-18) M). These PNA-PANI/Au electrodes can be utilized for detection of hybridization with the complementary sequence in 5 min sonicated M. tuberculosis genomic DNA within 1 min of hybridization time. These DNA-PANI/Au and PNA-PANI/Au electrodes can be used 6-7 and 13-15 times, respectively.