FRET-based dual-labeled oligonucleotide probes detect target DNA by probing a minor groove environment.

A novel amino-modifier ESF nucleoside AM37zA (1) containing a trifluoroacetyl (TFA)-protected amino group is designed for the functionalization of ODN probes after oligonucleotide synthesis. AM37zA (1) showed remarkable solvatochromicity and ODN probes containing deprotected AM37zA [ODN(N37zA)] discriminated single base alteration in target DNA based on changes in the fluorescence wavelength and intensity. Using AM37zA (1), a FRET-based dual-labeled ODN probe, ODN(F37zA), which contains two fluorophores at the major and minor groove sides of DNA, has been synthesized. The ODN(F37zA) incorporated with fluorescein by post-synthetic modification discriminated the target DNA sequence by a distinct change in the fluorescence intensity (S/N ratio of 20 : 1). Thus, an amino-modifier, AM37zA, and a FRET-based ODN probe, ODN(F37zA), are valuable for monitoring the microenvironment around nucleic acids and can act as powerful tools for examining the microstructures of DNA/RNA, including gene detection.

A fluorescent 3,7-bis-(naphthalen-1-ylethynylated)-2'-deoxyadenosine analogue reports thymidine in complementary DNA by a large emission Stokes shift.

The new environmentally responsive fluorescent nucleosides, 3,7-bis-(naphthalen-1-ylethynyl)-8-aza-3,7-dideaza-2'-deoxyadenosine (3n7nzA, 1) and 7-(naphthalen-1-ylethynyl)-8-aza-3,7-dideaza-2'-deoxyadenosine (37nzA, 2), have been synthesized. Both 3n7nzA (1) and 37nzA (2) possess large π-conjugated systems which extend into both the minor and major grooves or the major groove alone, respectively. The nucleosides exhibited large solvatochromic shifts (3n7nzA: Δλ = 45 nm, 37nzA: Δλ = 78 nm) and were examined for their ability to fluorimetrically report hybridization events. When incorporated into ODN probes, the bis-substituted 3n7nzA (1) selectively recognized thymidine on target strands which was reported by a distinct change in its emission wavelength in the long wavelength region, whereas 37nzA (2) showed a preference for pairing to cytidine and a smaller wavelength shift. Thus, 3n7nzA (1) has the potential for use as a fluorescent probe for structural studies of DNAs/RNAs including the detection of single-base alterations in target DNA sequences.

The fluorescently responsive 3-(naphthalen-1-ylethynyl)-3-deaza-2'-deoxyguanosine discriminates cytidine via the DNA minor groove.

A new environmentally responsive fluorescent nucleoside, 3-(naphthalen-1-ylethynyl)-3-deaza-2'-deoxyguanosine (3nzG), has been synthesized. The nucleoside, 3nzG, exhibited solvatochromic properties and when introduced into ODN probes it was able to recognize 2'-deoxycytidine in target strands by a distinct change in its emission wavelength through probing microenvironmental changes in the DNA minor groove. Thus, 3nzG has the potential for use as a fluorescent probe molecule for micro-structural studies of nucleic acids including the detection of single-base alterations in target DNA sequences.

Design and synthesis of environmentally sensitive fluorescent 2-naphthylethynylated 2'-deoxyadenosines: Detection of target DNA via changes in fluorescence intensity and wavelength.

Various C2-naphthylethynylated 2'-deoxyadenosines were synthesized as environmentally sensitive fluorescent (ESF) nucleosides and their photophysical properties were examined. Among the ESF nucleosides synthesized, four exhibited strong solvatochromicity, two of which were incorporated into oligodeoxynucleotides (ODNs). These ODN probes were able to detect target DNA through distinct changes in fluorescence intensity and wavelength and acted as effective reporter probes.

Design and synthesis of a novel fluorescent benzo[g]imidazo[4,5-c]quinoline nucleoside for monitoring base-pair-induced protonation with cytosine: distinguishing cytosine via changes in the intensity and wavelength of fluorescence.

A novel fluorescent benzo[g]imidazo[4,5-c]quinoline nucleoside (BIQ)A (1) comprising a 3-deaza-2'-deoxyadenosine skeleton was developed and used to monitor (BIQ)A-C base-pair formation in oligodeoxynucleotide (ODN) duplexes. The newly synthesized (BIQ)A exhibited distinct photophysical properties associated with its protonated/deprotonated forms (monomer: pKa 6.2) via dramatic changes in its absorption and fluorescence spectra. In ODN duplexes, the induced protonation of (BIQ)A occurred, even under alkalescent conditions when cytosine was the opposite base on the complementary strand; the resulting (BIQ)A-C base pairs were stable. By monitoring the protonation of (BIQ)A under neutral and alkalescent conditions, we could clearly discriminate cytosine through spectral changes in absorption and fluorescence. Similarly, we found that the demonstrated 3-deaza-2'-deoxyadenosine (3z)A forms a stable base pair with cytosine via N(1) protonation in ODN duplexes under neutral and acidic conditions (pH < 7.0). At lower pH values, (3z)A-containing ODNs could clearly discriminate cytosine through melting temperature (Tm) measurements. Therefore, ODN probes containing indicator nucleosides, such as (BIQ)A and (3z)A, exhibit great potential as bioprobes for genetic analysis and structural studies of nucleic acids.

Synthesis of 8-aza-3,7-dideaza-2'-deoxyadenosines possessing a new adenosine skeleton as an environmentally sensitive fluorescent nucleoside for monitoring the DNA minor groove.

8-Aza-3,7-dideaza-2'-deoxyadenosine 1 and its C3-naphthylethynylated derivative (3n7z)A (2) comprising a 8-aza-3,7-dideazapurine (pyrazolo[4,3-c]pyridine) skeleton were synthesized for the first time. In particular, nucleoside (3n7z)A (2) exhibited environmentally sensitive intramolecular charge transfer (ICT) emission because of electron transition in the coplanar conformer formed by nucleobase and naphthalene moieties. Its incorporation into oligodeoxynucleotide (ODN) probes enable a clear identification of a perfectly matched thymine (T) in the complementary strand by a distinct change in the emission wavelength. In addition, the fluorescence emission of the duplexes containing a cytosine/guanine (C/G) base pair flanking (3n7z)A (2) was strongly quenched by guanine only when the opposite base of the modified nucleoside was mismatched, enhancing its base identification ability. Thus, ODN probes containing (3n7z)A (2) acted as effective reporter probes for homogeneous single nucleotide polymorphism (SNP) typing.

Synthesis and photophysical properties of pyrene-labeled 3-deaza-2'-deoxyadenosines comprising a non-π-conjugated linker: fluorescence quenching-based oligodeoxynucleotide probes for thymine identification.

Pyrene-labeled 3-deaza-2'-deoxyadenosine comprising a non-π-conjugated linker (py3z)A (1) was synthesized and its photophysical properties were investigated. Oligodeoxynucleotide (ODN) probes containing (py3z)A (1) exhibited remarkable fluorescence quenching only when the opposite base of the complementary strand was the perfectly matched thymine. Such fluorescence quenching-based ODN probes exhibited excellent on-off switching properties, making them useful tools for single nucleotide polymorphism (SNP) genotyping and for the identification of target genes and structural studies of nucleic acids.

Molecular design of an environmentally sensitive fluorescent nucleoside, 3-deaza-2'-deoxyadenosine derivative: distinguishing thymine by probing the DNA minor groove.

An environmentally sensitive fluorescent nucleoside containing a 3-deazaadenine skeleton has been developed, and its photophysical properties were investigated. Newly developed C3-naphthylethynylated 3-deaza-2'-deoxyadenosine ((3nz) A, 1) exhibited dual fluorescence emission from an intramolecular charge-transfer state and a locally excited state, depending upon molecular coplanarity. DNA probes containing 1 clearly discriminated a perfectly matched thymine base on the complementary strand by a distinct change in emission wavelength.

Design of an environmentally sensitive fluorescent 8-aza-7-deaza-2'-deoxyadenosine derivative with dual fluorescence for the specific detection of thymine.

A novel environmentally sensitive fluorescent (ESF) purine nucleoside, (cna)A, was synthesized and its photophysical properties were investigated. The base-modified fluorescent nucleoside (cna)A exhibited remarkable solvatochromicity and environmentally sensitive dual fluorescence. By using (cna)A, we have developed highly thymine (T) selective fluorescent DNA probes that can sense the corresponding T in a target DNA/RNA sequence, as measured by a distinct change in emission wavelength.

Naphthalene-based environmentally sensitive fluorescent 8-substituted 2'-deoxyadenosines: application to DNA detection.

We synthesized various C8-naphthylethynylated 2'-deoxyadenosine derivatives and investigated their photophysical properties. Among them, cyano- and N,N-dimethylamino-substituted 8-naphthylethynylated 2'-deoxyadenosine derivatives ((cn)A and (dn)A) showed strong fluorescence with high quantum yields and a remarkable solvatofuorochromicity. In particular, fluorescence of N,N-dimethylamino-substituted (2,6dn)A was not quenched by neighboring guanines (Gs) when incorporated in DNA duplexes, in contrast to (cn)A. We developed a new fluorescent probe containing (2,6dn)A that can be used for the detection of target DNA via a bulge formation regardless of the neighboring sequences.

Enhancement of fluorescence quenching and exciplex formation in DNA major groove by double incorporation of modified fluorescent deoxyuridines.

5-(1-Naphthalenylethynyl)-2'-deoxyuridine ((N)U) and 5-[(4-cyano-1-naphthalenyl)ethynyl]-2'-deoxyuridine ((CN)U) were synthesized and incorporated into oligodeoxynucleotides. Fluorescence emissions of modified duplexes containing double (N)U were efficiently quenched depending upon the sequence pattern of the naphthalenes in DNA major groove, as compared to the duplex possessing single (N)U. When one of the naphthalene moieties has a cyano substituent, the exciplex emission from the chromophores in DNA major groove was observed at longer wavelength.

Fluorometric detection of adenine in target DNA by exciplex formation with fluorescent 8-arylethynylated deoxyguanosine.

We demonstrated an intriguing method to discriminate adenine by incident appearance of an intense new emission via exciplex formation in hybridization of target DNA with newly designed fluorescent 8-arylethynylated deoxyguanosine derivatives. We described the synthesis of such highly electron donating fluorescent guanosine derivatives and their incorporation into DNA oligomers which may be used for the structural study and the fluorometric analysis of nucleic acids.

Fluorescent nucleosides with 'on-off' switching function, pH-responsive fluorescent uridine derivatives.

We synthesized various pH-responsive fluorescent deoxyuridine derivatives (1a-g). These fluorescent nucleosides exhibited distinctive fluorescence at 470-600 nm in aqueous solvents containing methanol only at acidic to neutral pH values. In particular, 1f exhibited strong fluorescence only at pH range of 3.1-7.2 with a pK(a) of 6.1. Such pH-sensitive fluorescent nucleosides can be used as 'on-off' fluorescence switch for monitoring pH change in biological systems, particularly for cancer cell detection.

Stabilization of DNA duplex by 2-substituted adenine as a minor groove modifier.

2-(1-Naphthalenylethynyl)-2'-deoxyadenosine ((N)A) was synthesized and incorporated into oligodeoxynucleotides. DNA duplexes containing newly designed 5'-(N)AT-3'/3'-T(N)A-5' base pairs are considerably stabilized than unmodified duplexes by stacking interaction of naphthalene rings in the narrow minor groove as characterized by a new emission at longer wavelength and exciton coupled CD signals.

Design and synthesis of highly solvatochromic fluorescent 2'-deoxyguanosine and 2'-deoxyadenosine analogs.

We synthesized various substituted 8-styryl-2'-deoxyguanosine and 8-styryl-2'-deoxyadenosine derivatives. Among them only acetyl substituted 8-styryl-2'-deoxyguanosine analog 5b showed a remarkable solvatochromicity (Δλ(max)(em)=91 nm),that is, strong fluorescence at 477 nm in 1,4-dioxane, but in methanol the fluorescence was red shifted to 558 nm with very low intensity. Such environmentally sensitive solvatochromic fluorescent guanosine analogs may be useful as a sensor for investigating interactions of DNA with DNA binding proteins.

Singly and doubly labeled base-discriminating fluorescent oligonucleotide probes containing oxo-pyrene chromophore.

We have developed new oxo-pyrene labeled fluorescent nucleoside, (Oxo-Py)U which showed a strong fluorescence dependency on solvent polarity at long wavelength. The designed singly and doubly (Oxo-Py)U labeled fluorescent oligonucleotide probes were found highly efficient for the discrimination of A and consecutive AA bases of target DNA opposite to the labeled base via generation of enhanced fluorescence signal.

Design of environmentally sensitive fluorescent 2'-deoxyguanosine containing arylethynyl moieties: distinction of thymine base by base-discriminating fluorescent (BDF) probe.

We have synthesized various substituted 8-arylethynylated 2'-deoxyguanosine derivatives. Among them, acetyl substituted deoxyguanosine analogue 4c showed a remarkable solvent dependent fluorescence property, that is, an intense fluorescence in non-polar solvents but extremely weak fluorescence in polar solvents like methanol. By using solvatofluorochromic deoxyguanosine analogue 4c, we have developed highly thymine (T) selective fluorescent DNA probes that can sense T opposite 4c in a target DNA regardless of the flanking sequences. We were able to demonstrate that 4c can be used as a T specific base-discriminating fluorescent (BDF) nucleoside in homogeneous fluorescence assay.

Pyrene-labeled deoxyguanosine as a fluorescence sensor to discriminate single and double stranded DNA structures: design of ends free molecular beacons.

A novel fluorescent DNA probe containing pyrene-labeled C8 alkylamino-substituted 2'-deoxyguanosine was designed in order to discriminate single stranded and double stranded regions in DNA. This fluorescent sensor was used for the design of practically useful 3'- and 5'-ends free self-quenched molecular beacon (MB). Unique MB detectable by pyrene excimer fluorescence was also demonstrated.

Patient-specific design process and evaluation of a hip prosthesis femoral stem.

INTRODUCTION: There are several commercially available hip implant systems. However, for some cases, custom implant designed based on patient-specific anatomy can offer the patient the best available implant solution. Currently, there is a growing trend toward personalization of medical implants involving additive manufacturing into orthopedic medical implants' manufacturing. METHODS: This article introduces a systematic design methodology of femoral stem prosthesis based on patient's computer tomography data. Finite element analysis is used to evaluate and compare the micromotion and stress distribution of the customized femoral component and a conventional stem. RESULTS: The proposed customized femoral stem achieved close geometrical fit and fill between femoral canal and stem surfaces. The customized stem demonstrated lower micromotion (peak: 21 µm) than conventional stem (peak: 34 µm). Stress results indicate up to 89% increase in load transfer by conventional stem than custom stem because the higher stiffness of patient-specific femoral stem proximally increases the custom stem shielding in Gruen's zone 7. Moreover, patient-specific femoral stem transfers the load widely in metaphyseal region. CONCLUSION: The customized femoral stem presented satisfactory results related to primary stability, but compromising proximo-medial load transfer due to increased stem cross-sectional area increased stem stiffness.

6-Phenylpyrrolocytidine: An Intrinsically Fluorescent, Environmentally Responsive Nucleoside Analogue.

The detailed synthetic protocols for the preparation of phosphoramidite reagents compatible with standard, automated oligonucleotide synthesis for the 2'-deoxy- and ribo-6-phenylpyrrolocyitidine are reported. Each protocol starts with the parent nucleoside and prepares the 5'-O-dimethoxytrityl-N4 -benzoyl-5-iodocytosine derivative for the nucleobase modification chemistry. The key step is the direct formation of 6-phenylpyrrolocytosine aglycon via a sequential, one-pot Pd-catalyzed Sonogashira-type cross- coupling followed by a 5-endo-dig cyclization. Subsequent standard transformations provide the deoxy- and 2'-O-tert-butyldimethysilyl protected ribo- nucleoside phosphoramidite reagents. © 2019 by John Wiley & Sons, Inc.