Facile Quantification of Lesions Derived from 2‘-Deoxyguanosine in DNA

OxodG and Fapy•dG are two frequently formed DNA lesions that affect replication in vitro and in cells. They are also potentially important biomarkers for determining the effects of oxidative stress and aging in cells. We report the first method that enables one to selectively detect and individually quantify Fapy•dG and OxodG in DNA. The method relies upon selective chemical trapping of oxidized forms of the lesions with a biotinylated derivative of spermine. Selectivity for OxodG over Fapy•dG is achieved by varying the oxidant. The covalently tagged lesions are quantified using a fluorescence assay that is carried out in microtiter plates. The fluorescence assay is generally applicable to quantifying DNA lesions that can be tagged with biotin

Neomycin−Neomycin Dimer: An All-Carbohydrate Scaffold with High Affinity for AT-Rich DNA Duplexes

A dimeric neomycin−neomycin conjugate 3 with a flexible linker, 2,2′-(ethylenedioxy)bis(ethylamine), has been synthesized and characterized. Dimer 3 can selectively bind to AT-rich DNA duplexes with high affinity. Biophysical studies have been performed between 3 and different nucleic acids with varying base composition and conformation by using ITC (isothermal calorimetry), CD (circular dichroism), FID (fluorescent intercalator displacement), and UV (ultraviolet) thermal denaturation experiments. A few conclusions can be drawn from this study: (1) FID assay with 3 and polynucleotides demonstrates the preference of 3 toward AT-rich sequences over GC-rich sequences. (2) FID assay and UV thermal denaturation experiments show that 3 has a higher affinity for the poly(dA)·poly(dT) DNA duplex than for the poly(dA)·2poly(dT) DNA triplex. Contrary to neomycin, 3 destabilizes poly(dA)·2poly(dT) triplex but stabilizes poly(dA)·poly(dT) duplex, suggesting the major groove as the binding site. (3) UV thermal denaturation studies and ITC experiments show that 3 stabilizes continuous AT-tract DNA better than DNA duplexes with alternating AT bases. (4) CD and FID titration studies show a DNA binding site size of 10−12 base pairs/drug, depending upon the structure/sequence of the duplex for AT-rich DNA duplexes. (5) FID and ITC titration between 3 and an intramolecular DNA duplex [d(5′-A12-x-T12-3′), x = hexaethylene glycol linker] results in a binding stoichiometry of 1:1 with a binding constant ∼108 M−1 at 100 mM KCl. (6) FID assay using 3 and 512 hairpin DNA sequences that vary in their AT base content and placement also show a higher binding selectivity of 3 toward continuous AT-rich than toward DNA duplexes with alternate AT base pairs. (7) Salt-dependent studies indicate the formation of three ion pairs during binding of the DNA duplex d[5′-A12-x-T12-3′] and 3. (8) ITC-derived binding constants between 3 and DNA duplexes have the following order: AT continuous, d[5′-G3A5T5C3-3′] > AT alternate, d[5′-G3(AT)5C3-3′] > GC-rich d[5′-A3G5C5T3-3′]. (9) 3 binds to the AT-tract-containing DNA duplex (B* DNA, d[5′-G3A5T5C3-3′]) with 1 order of magnitude higher affinity than to a DNA duplex with alternating AT base pairs (B DNA, d[5′-G3(AT)5C3-3′]) and with almost 3 orders of magnitude higher affinity than a GC-rich DNA (A-form, d[5′-A3G5C5T3-3′])

Combining the Best in Triplex Recognition: Synthesis and Nucleic Acid Binding of a BQQ−Neomycin Conjugate

Synthesis of a BQQ−neomycin conjugate is reported. The conjugate combines two ligands, one known to intercalate triplexes (BQQ) and another known to bind in the triplex groove (neomycin). The conjugate stabilizes T·A·T, as well as mixed base DNA triplex, better than neomycin, BQQ, or a combination of both. The conjugate selectively stabilizes the triplex (in the presence of physiological salt concentrations), with as little as 4 μM of the ligand leading to a ΔTm of >60 °C. Competition dialysis studies show a clear preference for the drug binding to triplex DNA/RNA over the duplex/single strand structures. Modeling studies suggest a structure of neomycin bound to the larger W−H (Watson−Hoogsteen) groove with BQQ intercalated between the triplex bases

Enzymatic Ring-Opening Polymerization of Trimethylene Carbonate with Macrodiol: Synthesis of Block Poly(ester-<i>co</i>-carbonate) for Biomaterial Preparation

Novozym 435-catalyzed ring-opening polymerization (ROP) of trimethylene carbonate (TMC) with telechelic hydroxylated poly[(R)-3-hydroxybutyrate] [PHB-diol; Mn = 3000 g/mol (GPC)] as initiator gave di-block poly(HB-co-TMC) with different weight percents of the blocks and Mn of 4400–8700 g/mol (GPC) in 54–89% yield, being the first enzymatic preparation of block poly(ester-co-carbonate). The generality of the novel enzymatic method was demonstrated by the enzymatic ROP of TMC with poly(ε-caprolactone)-diol [PCL-diol; Mn of 4200 g/mol (GPC)] to give A–B–A tri-block-poly(TMC-co-CL-co-TMC) with different weight percents of the blocks and Mn of 7700–10600 g/mol (GPC) in 54–67% yield. The prepared block poly(ester-co-carbonate) with two terminal hydroxyl groups was proven to be a useful starting material for the further preparation of thermoplastic block copolymers. Polymerization of di-block poly(24 wt % HB-co-76 wt % TMC) with methylene diphenyl-4,4′-diisocyanate (MDI) afforded the corresponding polyurethane with Mn of 53 800 g/mol (GPC) in 94% yield. The polymer showed excellent thermoplastic properties (Tm of 144 and 152 °C, Tg of −9 °C, εb of 252%, σmax of 6.37 MPa, and E of 23 MPa), being potentially useful for soft tissue engineering

Aminoglycoside (Neomycin) Preference Is for A-Form Nucleic Acids, Not Just RNA: Results from a Competition Dialysis Study

Aminoglycosides have been at the forefront of antimicrobial therapy for the past 50 years. Their specificity is believed to lie in binding duplex RNAs (rRNA). Competition dialysis studies of various nucleic acid forms with 9-aminoacridine, quinacrine, and a neomycin-acridine conjugate were carried out. Our results suggest a strong preference for aminoglycoside binding to nucleic acids that can adopt an A-type conformation. These results challenge the common belief that aminoglycoside specificity is simply for duplex RNAs

Synthesis and Spectroscopic Studies of the Aminoglycoside (Neomycin)−Perylene Conjugate Binding to Human Telomeric DNA

Synthesis of a novel perylene−neomycin conjugate (3) and the properties of its binding to human telomeric G-quadruplex DNA, 5′-d[AG3(T2AG3)3] (4), are reported. Various spectroscopic techniques were employed to characterize the binding of conjugate 3 to 4. A competition dialysis assay revealed that 3 preferentially binds to 4, in the presence of other nucleic acids, including DNA, RNA, DNA−RNA hybrids, and other higher-order structures (single strands, duplexes, triplexes, other G-quadruplexes, and the i-motif). UV thermal denaturation studies showed that thermal stabilization of 4 increases as a function of the increasing concentration of 3. The fluorescence intercalator displacement (FID) assay displayed a significantly tighter binding of 3 with 4 as compared to its parent constituents [220-fold stronger than neomycin (1) and 4.5-fold stronger than perylene diamine (2), respectively]. The binding of 3 with 4 resulted in pronounced changes in the molar ellipticity of the DNA absorption region as confirmed by circular dichroism. The UV−vis absorption studies of the binding of 3 to 4 resulted in a red shift in the spectrum of 3 as well as a marked hypochromic change in the perylene absorption region, suggesting that the ligand−quadruplex interaction involves stacking of the perylene moiety. Docking studies suggest that the perylene moiety serves as a bridge that end stacks on 4, making contacts with two thymine bases in the loop, while the two neomycin moieties branch into the grooves of 4

miR-140 suppresses tumor growth and metastasis of NSCLC in nude mice.

<p>(A) photography of tumors formed. (B) growth curve drawn by measuring tumor volumes on the indicated days. (C) representative H&E-stained sections of the lung tissues isolated from mice (100×). The total numbers of metastatic lesions in the lungs were counted. **<i>P</i><0.01.</p

miR-140 suppresses NSCLC cell migration and invasion in vitro.

<p>The wound healing assays (A) and invasion assays (B) of A549 and H157 cells infected with miR-140 or miR-control lentivirus. The invasion assays were determined using Transwell assays with Matrigel. Magnification: 100×. **<i>P</i><0.01.</p

miR-140 Suppresses Tumor Growth and Metastasis of Non-Small Cell Lung Cancer by Targeting Insulin-Like Growth Factor 1 Receptor

<div><p>MicroRNAs (miRNAs) are a class of small noncoding RNA molecules that play important roles in carcinogenesis and tumor progression. In this study, we investigated the roles and mechanisms of miR-140 in human non-small cell lung cancer (NSCLC). We found that miR-140 is significantly downregulated in NSCLC tissues and cell lines. Both gain-of-function and loss-of-function studies demonstrated that miR-140 suppresses NSCLC cell proliferation, migration, and invasion in vitro. Importantly, overexpression of miR-140 effectively repressed tumor growth and metastasis in nude mouse models. Integrated analysis identified IGF1R as a direct and functional target of miR-140. Knockdown of IGF1R inhibited cell proliferation and invasion resembling that of miR-140 overexpression, while overexpression of IGF1R attenuated the function of miR-140 in NSCLC cells. Together, our results highlight the significance of miR-140 and IGF1R in the development and progression of NSCLC.</p> </div

IGF1R is involved in miR-140-induced growth and invasion inhibition in A549 cells.

<p>A549 cells were infected with specific si IGF1R, or transfected with IGF1R plasmid lacking 3’ UTR along with miR-140. (A) Western blotting analysis. (B) cell viability assay (CCK-8). (C) cell cycle analysis. (D) cell apoptosis assays. (E) Transwell invasion assays. *<i>P</i><0.05, **<i>P</i><0.01.</p