Design and synthesis of a photocleavable fluorescent nucleotide 3'-O-allyl-dGTP-PC-Bodipy-FL-510 as a reversible terminator for DNA sequencing by synthesis

DNA sequencing by synthesis (SBS) using reversible fluorescent nucleotide terminators is potentially an efficient approach to address the limitations of current DNA sequencing techniques. Here, we report the design and synthesis of a 3'-O-allyl photocleavable fluorescent nucleotide analogue, 3'-O-allyl-dGTP-PC-Bodipy-FL-510, as a reversible terminator for SBS. The nucleotide is efficiently incorporated by DNA polymerase into a growing DNA strand to terminate the polymerase reaction. After that, the fluorophore is photocleaved quantitatively by irradiation at 355 nm, and the allyl group is rapidly and efficiently removed by using a Pd-catalyzed reaction under DNA-compatible conditions to regenerate a free 3'-OH group to reinitiate the polymerase reaction. Two cycles of such steps were successfully demonstrated to sequence a homopolymeric region of a DNA template, facilitating the development of SBS as a viable approach for high-throughput DNA sequencing.     

DNA as a template for synthesis of fluorescent gold nanoclusters

Water-soluble blue-emitting gold nanoclusters have been synthesized using dsDNA as a template without any additional reducing agent. The features of the formed nanoclusters have been revealed by fluorescence and electronic absorption spectroscopy as well as transmission electron microscopy. The prepared gold nanoclusters have been highly stable at physiological pH without any further modification.     

A cleavable amino-thiol linker for reversible linking of amines to DNA

A cleavable heterobifunctional cross-linker for the reversible conjugation of amines to thiol-modified DNA has been developed and tested. The succinimidyl 2-(vinylsulfonyl)ethyl carbonate (SVEC) was prepared in three steps and tested for its ability to react with amines and thiols. The linker was efficient for binding leucine to a thiol-modified DNA sequence and for releasing the amino acid at pH 11.8.     

Fluorescent detection of single nucleotide polymorphism utilizing a hairpin DNA containing a nucleotide base analog pyrrolo-deoxycytidine as a fluorescent probe

A novel fluorescent method for the detection of single nucleotide polymorphism (SNP) was developed using a hairpin DNA containing nucleotide base analog pyrrolo-deoxycytidine (P-dC) as a fluorescent probe. This fluorescent probe was designed by incorporating a fluorescent P-dC into a stem of the hairpin DNA, whose sequence of the loop moiety complemented the target single strand DNA (ss-DNA). In the absence of the target ss-DNA, the fluorescent probe stays a closed configuration in which the P-dC is located in the double strand stem of the fluorescent probe, such that there is weak fluorescence, attributed to a more efficient stacking and collisional quenching of neighboring bases. In the presence of target ss-DNA, upon hybridizing the ss-DNA to the loop moiety, a stem-loop of the fluorescent probe is opened and the P-dC is located in the ss-DNA, thus resulting in strong fluorescence. The effective discrimination of the SNP, including single base mismatch ss-DNA (A, T, G) and double mismatch DNA (C, C), against perfect complementary ss-DNA was achieved by increased fluorescence intensity, and verified by thermal denaturation and circular dichroism spectroscopy. Relative fluorescence intensity had a linear relationship with the concentration of perfect complementary ss-DNA and ranged from 50 nM to 3.0 μM. The linear regression equation was F/F₀ = 2.73 C (μM) + 1.14 (R = 0.9961) and the detection limit of perfect complementary ss-DNA was 16 nM (S/N = 3). This study demonstrates that a hairpin DNA containing nucleotide base analog P-dC is a promising fluorescent probe for the effective discrimination of SNP and for highly sensitive detection of perfect complementary DNA.     

4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene as a bright fluorescent label for DNA

4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) as a fluorescent label can be incorporated into DNA by two conceptually different ways: the non-nucleosidic DNA base surrogate Bo exhibits high brightness but no preferential base-pairing properties, whereas the BODIPY-modified uridine BodU has reduced quantum yields but shows preferred Watson-Crick base pairing with adenine.     

The synthesis of a menthol derivative of 2-aminopurine as a fluorescent DNA lesion

An efficient synthetic route to the phosphoramidite of a menthol functionalized guanosine analog is presented. Two procedures were executed for the key introduction of the 6′-allyl menthyl moiety. Stille vinylation on 6-O-tosylguanosine followed by cross-metathesis using an excess of allyl menthyl ether proved to be less efficient than a Stille coupling on the same tosylate using an advanced menthyl-allyl stannane derivative. Incorporation of the modified nucleoside using the phosphoramidite method into a DNA 50-mer proceeded uneventfully.     

Design and synthesis of a new fluorescent tripod for chemosensor applications

A new yellow-green fluorescent tripod based 1,8-naphthalimide has been synthesized and characterised. Its photophysical properties have been investigated in organic solvents of different polarity. The effect that the metal ions (Cd²⁺, Co²⁺, Zn²⁺, Mn²⁺, Mg²⁺, Ni²⁺, Pb²⁺, Cu²⁺, Ba²⁺, Fe³⁺ and Ag⁺) produce upon the fluorescent intensity of acetonitrile solutions of the tripod has been discussed viewing its potential applications as a detector for metal cations. The influence of protons on the fluorescence intensity of the tripod in DMF and methanol–water (1:4 v/v) solutions has also been investigated.     

Design of a new fluorescent cofactor for DNA methyltransferases and sequence-specific labeling of DNA

Sequence-specific labeling of DNA is of immense interest for analytical and functional studies of DNA. We present a novel approach for sequence-specific labeling of DNA using a newly designed fluorescent cofactor for the DNA methyltransferase from Thermus aquaticus (M.TaqI). Naturally, M.TaqI catalyzes the nucleophilic attack of the exocyclic amino group of adenine within the double-stranded 5'-TCGA-3' DNA sequence onto the methyl group of the cofactor S-adenosyl-L-methionine (AdoMet) leading to methyl group transfer. The design of a new fluorescent cofactor for covalent labeling of DNA was based on three criteria: (1) Replacement of the methionine side chain of the natural cofactor AdoMet by an aziridinyl residue leads to M.TaqI-catalyzed nucleophilic ring opening and coupling of the whole nucleoside to DNA. (2) The adenosyl moiety is the molecular anchor for cofactor binding. (3) Attachment of a fluorophore via a flexible linker to the 8-position of the adenosyl moiety does not block cofactor binding. According to these criteria the new fluorescent cofactor 8-amino[1'-(N'-dansyl)-4'-aminobutyl]-5'-(1-aziridinyl)-5'-deoxyadenosine (3) was synthesized. 3 binds about 4-fold better than the natural cofactor AdoMet to M.TaqI and is coupled with a short duplex oligodeoxynucleotide by M.TaqI. The identity of the expected modified nucleoside was verified by electrospray ionization mass spectrometry after enzymatic fragmentation of the product duplex. In addition, the new cofactor 3 was used to sequence-specifically label plasmid DNA in a M.TaqI-catalyzed reaction.     

Design and synthesis of a solid-phase fluorescent mass tag

In this study, we demonstrate the design of a new solid-phase fluorescent mass tag (FMT) that contains the following features: (1) the FMT is synthesized using Fmoc chemistry which is simple, rapid, and cost-effective; (2) lysine is used as a uniformly labeled amino acid (using stable isotopes) to allow 8 Da difference between "heavy" and "light" tags; (3) a fluorescent molecule is coupled to the isotope tag that allows a tagged peptide to be detected by online fluorescence; and (4) an iodoacetyl reactive group provides cysteine reactivity. Using MALDI-TOF MS and HPLC, we show that the FMT reagent can be used to label standard cysteine-containing peptides as well as cysteine-containing peptides from a BSA tryptic digest.     

Single base interrogation by a fluorescent nucleotide: each of the four DNA bases identified by fluorescence spectroscopy

Nucleoside C, which contains a rigid nitroxide spin label, is effectively reduced in DNA by sodium sulfide to the corresponding amine, yielding a fluorescent probe (Cf) that can report the identity of its base-pairing partner in duplex DNA.     

Design, synthesis and evaluation of a fluorescent peptidyl sensor for the selective recognition of arsenite

A series of fluorescently labelled phytochelatin-like peptide sequences has been designed and synthesised as molecules with the ability both to bind to arsenite and to report this binding event. The peptides have been evaluated against a panel of biologically significant ions. The results of these initial studies are reported herein, and a structure which displays selectivity for As(III) over As(V) and other anions has been identified.     

Comb-like copolymers as self-coating,low-viscosity and high-resolution matrices for DNA sequencing

Comb-like copolymers with a polyacrylamide backbone and poly(N,N-dimethylacrylamide) grafts were prepared, as a way to combine the superior sieving properties of polyacrylamide with the self-coating properties of polydimethylacrylamide. These matrices function well in the absence of a capillary coating, and achieve separation performances for single-stranded DNA that are comparable to those of state-of-the-art long-chain linear polyacrylamide. Structural parameters such as the grafting density and the polymer molecular mass were varied, and good performance appears to be achieved with a relatively large range of parameters. Surprisingly, excellent separation is achieved even with matrices that have a viscosity as low as 200 mPa/s. A discussion of the physics underlying this behavior is provided.     

Design and synthesis of fluorescent galactolipid probes

Fluorescent galactolipid analogues (1, 1a, and 2) have been synthesized with a pyrene group attached to the amino-terminal of a hexanoyl chain bound to an otherwise normal galactolipid structure. The synthetic lipids are obtained from peracetyl galactose by a general and versatile procedure based on the trichloroacetimidate methodology. The intense violet fluorescence (376 and 395 nm) and good photostability of pyrene make these compounds highly suitable as probes to study galactolipid metabolism. As proof of concept, we report that compound 2 is a valid tool to detect galactolipase activity in enzymatic preparations of potato tubers.     

High-sensitive fluorescent DNA sequencing and its application for detection and mass-screening of point mutations.

We describe a rapid and sensitive DNA sequencing method for an automated fluorescent DNA sequencer (AFDS) and its application for detection of point mutations. The method is based on an improved cycle sequencing procedure in which only 10-50 fmol of template DNA is required. Furthermore, it is able to use crude DNA preparation as a template as well as the purified one. Thus, the improved method provided a simplified procedure for sequencing of various types of DNA, including cosmid DNA, in which purification steps were unnecessary. We also developed a novel system for detection of point mutations using AFDS. A set of four lanes is used for the parallel analysis of single-base profiles of four different samples, instead of for the four-base profile of a sample. The AFDS exhibits the base profiles of the samples with four different colors in the analyzed data, which enables us to identify a mutation as an additional peak with a color specific for the lane. The feasibility of our system was tested by analyzing polymerase chain reaction (PCR)-amplified genomic DNAs from four individuals including a carrier of a mutation of C to T. The mutation was clearly identified as an additional "T" peak of a color specific for the carrier. The mutation was also detectable even if 16 individuals including the carrier were simultaneously analyzed on a set of four lanes (four individual samples for each lane). Thus, the novel system is useful for simultaneous detection of mutations in a large number of individual samples.     

Design, synthesis and characterization of a novel fluorescent probe for nitric oxide based on difluoroboradiaza-s-indacene fluorophore

Nitric oxide (NO) is a gaseous, free radical, which plays a role as an intracellular second messenger and a diffusible intercellular messenger. To obtain evidence for NO function in vivo, 1,3,5,7-tetramethyl-8-(4'-aminophenyl-N-(2'-amino)-phenzyl)-difluoroboradiaza-s-indacene (TMAPABODIPY) was designed and synthesized as a fluorescent probe for nitric oxide, which features high photostability and no pH dependency over a wide pH range. The fluorescence of TMAPABODIPY itself is very strong. When TMAPABODIPY traps NO in the presence of dioxygen, the weak fluorescent triazole form is obtained, which offers the advantages of specificity, and sensitivity for direct detection of NO. The relationship was obtained between the quenching fluorescence intensity and NO concentration in the range 0.02-4.0 micromol l(-1). The detection limit is 5 nmol l(-1) (S/N = 3). The proposed method has been used to monitor the release of NO from S-nitrosocysteine, a NO-releasing agent.     

Disulfide bond as a cleavable linker for molecular self-assembly and hydrogelation

Here we reported on the first example of using the disulfide bond as a cleavable linker to control molecular self-assembly and the formation of small molecular (SM) hydrogels.     

Design and synthesis of a fluorescent reporter of protein kinase activity

There is widespread interest in developing fluorescent reporters of protein kinase activity, species that can furnish a visual readout of both when and where intracellular kinases are activated in response to a stimulus. We have constructed and identified, via a combination of rational design, library synthesis, and screening, a difluorofluorescein-appended peptide-based species that responds to protein kinase C phosphorylation in a fluorescently sensitive fashion. The phosphorylation-induced divalent metal ion-mediated 265% enhancement in fluorescence proceeds with a V(max) of 8.5 micromol/min.mg and a K(m) of 20.5 microM.     

A reversible fluorescent DNA logic gate based on graphene oxide and its application for iodide sensing

A simple and reliable fluorescent DNA logic gate is developed by utilizing graphene oxide as a signal transducer and mercury ions and iodide as mechanical activators.     

Design, synthesis and evaluation of near-infrared fluorescent pH indicators in a physiologically relevant range

Removal of a hydroxylsulfonylbutyl arm from indocyanine green dye produced a pH-sensitive near-infrared (NIR) fluorescent indicator that is useful at physiological range.