Cooperative Non-enzymatic Base Recognition and the Stability of the G-U Wobble Pair

INVESTIGATIONS of paired complementary polynucleotides containing occasional mismatched bases have indicated that these bases are looped out of the double helical regions thereby making the helix unstable. But in a system containing mismatched G and U bases, no such definite conclusion could be drawn^1–4—stoichiometry and T_m values of such complexes can be interpreted in terms of the formation of a G-U wobble pair⁴. Recently the complexes formed by self-complementary block oligomers interrupted by mismatched bases, for example, A_nXU_n (X=G, C and U), have been studied in detail⁵·⁶. It seems that stacking interactions play a much more dominant role in the stabilization of double helices than was previously thought. Thus the extent of looping out and of non-Watson-Crick base pairing can hardly be assessed independently.     

High Field 1H-NMR Analysis of the 1:1 Intercalation Complex of the Antitumor Agent Mitoxantrone and the DNA Duplex [d(CpGpCpG)]2

Abstract

Complete ¹H-nmr assignment has been achieved of the stoichiometric 1:1 complex of the antitumor agent mitoxantrone with the duplex oligomer [d(CpGpCpG)]₂. The techniques used included 2D-COSY, 1D-NOE and 2D-HH-INADEQUATE. Comparisons of ¹H and ¹³C chemical shift changes upon addition of drug suggest symmetrical intercalative binding to the center of the tetramer. NOE difference measurements and ³¹P studies suggest binding of the terminal OH groups of the side chains to the central phosphate groups such that the methylene groups are proximate to C(3)6, C(3)6 and G(4)8 base protons all in the major groove. The data suggest that the side chains bind to the neighboring base pairs from the intercalation site. This is in accord with independent evidence of G,C base preference for binding from spectroscopic and electron microscopy studies.     

Effects of deficient of the Hoogsteen base-pairs on the G-quadruplex stabilization and binding mode of a cationic porphyrin

BackgroundIn stabilization of the G-quadruplex, formation of a Hoogsteen base-pair between the guanine (G) bases is essential. However, the contribution of each Hoogsteen base-pair at different positions to whole stability of the G-quadruplex has not been known. In this study, the effect of a deficiency of the Hoogsteen type hydrogen bond in the G-quadruplex stability was investigated. Spectral properties of meso-tetrakis(1-methylpyridinium-4-yl)porphyrin (TMPyP) associated with various G-quadruplexes were also examined.MethodsThe thermal stability of the thrombin-binding DNA aptamer 5′G₁G₂TTG₅G₆TG₈TG₁₀G₁₁TTG₁₄G₁₅ G-quadruplex, in which the guanine (G) base at 1, 2, 5, 6 and 8th positions was replaced with an inosine (I) base, one at a time, was investigated by circular dichroism (CD). The absorption, CD and fluorescence decay curve for the G-quadruplex associated TMPyP were also measured.ResultsThe transition from the G-quadruplex to a single stranded form was endothermic and induced by an increase in entropy. The order in stability was 0>8>6>2>5>1, where the numbers denote the position of the replacement and 0 represents no replacements of the G base, suggesting the significant contribution of the G₁ base in the stability of the G-quadruplex. Alteration in the spectral property of TMPyP briefly followed the order in thermal stability.ConclusionsReplacement of a G base with an I base resulted in destabilization of the G-quadruplex. The missing hydrogen bond at position 1 destabilized the G-quadruplex most efficiently. TMPyP binds near the I base-replaced location namely, the side of the G-quadruplex.General significanceThe Hoogsteen base-pairing is confirmed to be essential in stabilization of G-quadruplex. When G is replaced with I, the latter base is mobile to interact with cationic porphyrin.     

Comparative Studies of the Thermodynamic Stabilities Between Sheared A:G and Watson-Crick A:U(T) Base Pairs in RNA and DNA

Abstract

Thermodynamic parameters for duplex formation were determined from CD melting curves for r(GGACGAGUCC)₂ and d(GGACGAGTCC)₂, both of which form two consecutive ‘sheared’ A:G base pairs at the center [Katahira et al. (1993) Nucleic Acids Res. 21, 5418–5424; Katahira et al., (1994) Nucleic Acids Res. 22, 2752–27591. The parameters were determined also for r(GGACUAGUCC)₂ and d(GGACTAGTCC)₂, where the A:G mismatches are replaced by Watson-Crick A:U(T) base pairs. Thermodynamic properties for duplex formation are compared between the sheared and the Watson-Crick base pairs, and between RNA and DNA. Difference in the thermodynamic stability is analyzed and discussed in terms of enthalpy and entropy changes. The characteristic features in CD spectra of RNA and DNA containing the sheared A:G base pairs are also reported.

     

Characterization of peripheral blood stem cell grafts mobilized by granulocyte colony-stimulating factor and plerixafor compared with granulocyte colony-stimulating factor alone

Background aimsThis study aimed to characterize the immune effectors contained in apheresis samples obtained from patients with grafts mobilized with plerixafor and granulocyte colony-stimulating factor (G-CSF) (P+G) compared with grafts mobilized with G-CSF alone (G).MethodsAliquots of apheresis samples were obtained from 36 patients with malignant diseases after mobilization with G (n = 18) or P+G (n = 18). The phenotype and cytokine secretion profile of T cell and dendritic cell subsets were characterized by multicolor cytometry including intracellular cytokine staining.ResultsIn grafts collected after mobilization with P+G, there was a significantly higher percentage of CD3⁺ T cells compared with samples collected after mobilization with G alone. On a functional level, a significant increase of interferon-γ and tumor necrosis factor-α secreting CD8⁺ T cells was observed in the P+G group compared with the G group. CD4⁺Foxp3⁺ regulatory T cells were similar in both groups but exhibited a lower expression of inducible costimulatory molecule and a significantly higher expression of CD127 in the P+G group. Myeloid dendritic cells (MDCs) and BDCA3⁺ dendritic cells were similar in both groups. In contrast, plasmacytoid dendritic cells (PDCs) (CD123⁺BDCA2⁺HLA-DR⁺) were significantly increased in the P+G grafts, leading to a higher PDC-to-MDC ratio. PDCs mobilized by P+G displayed different functional markers—a higher percentage of ILT7⁺ PDCs and decreased expression of CD86—suggesting a potential regulatory capacity of PDCs mobilized by P+G.ConclusionsGrafts mobilized with P+G exhibited major different functional features compared with grafts mobilized with G alone, suggesting that such grafts may have an impact on patient outcome after autologous stem cell transplantation.     

Solution Structure of a GAAG Tetraloop in Helix 6 of SRP RNA from Pyrococcus furiosus

The NMR structure of a 12-mer RNA derived from the helix 6 of SRP RNA from Pyrococcus furiosus, whose loop-closing base pair is U:G, was determined, and the structural and thermodynamic properties of the RNA were compared with those of a mutant RNA with the C:G closing base pair. Although the structures of the two RNAs are similar to each other and adopt the GNRR motif, the conformational stabilities are significantly different to each other. It was suggested that weaker stacking interaction of the GAAG loop with the U:G closing base pair in 12-mer RNA causes the lower conformational stability.     

Stacking of Crick Wobble pair and Watson-Crick pair: stability rules of G-U pairs at ends of helical stems in tRNAs and the relation to codon-anticodon Wobble interaction.

The occurrence of the noncomplementary G-U base pair at the end of a helix is found to be governed by stacking interactions. As a rule, a G-U pair with G on the 5'-side of a Watson-Crick base pair exhibits strikingly greater stacking overlap with the Watson-Crick base pair than a G-U pair on the 3'-side of a Watson-Crick base pair. The former arrangement is expected to be more stable and indeed is observed 29 times out of 32 in the known transfer RNA molecules. In accordance with this rule, the major wobble base pairs G-U or I-U in codon-anticodon interactions have G or I on the 5'-side of the anticodon. Similarly, in initiator tRNAs, this rule is obeyed where now the G is the first letter of the codon (5'-side). In the situation where U is in the wobble position of the anticodon, it is usually substituted at C(5) andmay also have a 2-thio group and it can read one to four codons depending on its modifications. A G at the wobble position of the anticodon can recognize the two codons ending with U or C and modification of G (unless it is I) does not change its reading properties.     

Unraveling stem cell and progenitor subsets in autologous grafts according to methods of mobilization: implications for prediction of hematopoietic recovery

Background aimsIn the autologous setting, granulocyte colony-stimulating factor (G-CSF) (G), or, when failing, G plus plerixafor (G+P), are common regimens for mobilization of stem cells into peripheral blood. To delineate mobilization effects on graft composition and hematopoietic recovery, we compared contents of stem cells and progenitor cells in products of G+P- and G patients. Paired samples of G+P patients and prior insufficient G mobilization were available for analyses.MethodsSubset analyses of grafts were performed by flow cytometry and myeloid colony-forming assay. In search of new markers to ascertain graft quality, we determined the fractions of aldehyde dehydrogenase bright (ALDH^br) cells.ResultsG grafts contained higher percentages of CD34+ cells, CD34+CD38- cells, and committed progenitors (CD34+CD38+) compared with G+P grafts. A detailed characterization of the mobilized CD34+ cell subset showed higher percentages of CD38– among the CD34+ cells of the G+P group (P = 0.032). In contrast, the CD34+ cell subset in G grafts was characterized by a higher percentage of ALDH^br cells (P < 0.0001). Studying engraftment and day +100 graft function the G and G+P transplanted patients were comparable with respect to neutrophils, whereas in platelets they differed. In the prediction of engraftment and hematopoietic recovery, the dose of infused ALDH^br cells correlated best to both platelet (r = 0.565, P = 0.002) and neutrophil reconstitution (r = 0.366, P = 0.06).ConclusionsBesides showing dissimilar distributions of CD34+CD38– cells and progenitors in G and G+P grafts, this study further designated ALDH^br as a promising marker in determination and prediction of graft quality and hematopoietic recovery.     

研究报告: 基于遗传算法的计算机辅助策略优化孕酮适配体

目的 本研究致力于优化孕酮（progesterone，P4）适配体的亲和力和选择性。方法 基于遗传算法（genetic algorithm，GA）的计算机辅助优化策略（in silico maturation，ISM），进行了4轮GA操作（含交叉变异、单点突变和双点突变操作），构建了初始文库和G1、G2、G3代ssDNA作为新的候选适配体库，采用分子对接对候选适配体进行筛选和分析，并使用迭代策略不断优化适配体。此外，还提出了一种较为准确预测ssDNA三级结构的方法，首先使用Mfold预测二级结构，继而使用RNAComposer建立与ssDNA相对应的RNA三级结构，输出的PDB文件使用Discovery Studio将RNA修改为DNA，最后使用Molecular Operating Environment对结构进行能量最小化处理。结果 到G2代，在局部搜索空间对P4S-0进行优化，筛选出P4G1-14、P4G2-20、P4G1-6、P4G1-7和P4G2-14这5条适配体作为P4的最佳候选适配体。采用AuNPs比色法初步验证优化后适配体的亲和力，继而构建了基于适配体结构开关的荧光法测定适配体的解离常数（equilibrium dissociation constant，K_D），并以此方法对适配体的选择性（对双酚A、雌二醇、睾酮和皮质醇）进行了评估。结论 通过ISM优化后的适配体，对P4的亲和力较原适配体有了较大提升，仍保留着识别结构类似分子的选择性。     

Synthesis of Amide Linked Nucleosides at the 6 Position of Deoxy Inosine and Their application to DNA synthesis,Hybridization Studies.

Abstract

Synthesis of amide linked nucleosides at the 6 position of the purine base to recognize “G:C” base pair in a DNA duplex is described. Here we describe the synthesis of amide linked nucleosides containing heteroatoms N, O and C and their application to DNA synthesis and hybridization studies.     

Structural Studies of DNA Fragments: The G·T Wobble Base Pair in A,B and Z DNA; The G·A Base Pair in B-DNA

Abstract

The crystal structures of five double helical DNA fragments containing non-Watson-Crick complementary base pairs are reviewed. They comprise four fragments containing G·T base pairs: two deoxyoctamers d(GGGGCTCC) and d(GGGGTCCC) which crystallise as A type helices; a deoxydodecamer d(CGCGAATTTGCG) which crystallises in the B-DNA conformation; and the deoxyhexamer d(TGCGCG), which crystallises as a Z-DNA helix. In all four duplexes the G and T bases form wobble base pairs, with bases in the major tautomer forms and hydrogen bonds linking N1 of G with 02 of T and 06 of G with N3 of T. The X-ray analyses establish that the G·T wobble base pair can be accommodated in the A, B or Z double helix with minimal distortion of the global conformation. There are, however, changes in base stacking in the neighbourhood of the mismatched bases. The fifth structure, d(CGCGAATTAGCG), contains the purine purine mismatch G·A where G is in the anti and A in the syn conformation. The results represent the first direct structure determinations of base pair mismatches in DNA fragments and are discussed in relation to the fidelity of replication and mismatch recognition.     

Variable Temperature Infrared Spectroscopy of Cytosine-Guanine Base Pairs: Tautomerism Versus Polarization

Abstract

This report describes an infrared (IR) spectroscopic study of a model cytosine - guanine base pair. This base pair is part of a self-consistent experimental system based on lipophilic ribose derivatives of cytidine (C), guanosine (G) and O⁶-methylguanosine (O⁶MeG) that are soluble in non-aqueous, low dielectric solvents at appreciable concentrations. Previous experiments on this system have revealed different rotation dynamics for the amino bonds within the CG base pair, an observation that could be explained by the presence of rare tautomers (P.O. Lowdin, Reviews of Modern Physics 35, 724 (1963)), or by mutual polarization of the base pairs (L.D. Williams, N.G. Williams and B.R. Shaw, J. Am. Chem. Soc. 112, 829 (1990)). The IR spectra in the OH and NH stretching region indicate formation of hydrogen-bonded CG base pairs and self associates in 1,2-dichlorobenzene over a temperature range from 10 to 290K. Changes in the lineshapes and intensities of the IR bands with temperature correlate with phase transitions of the solvent but no evidence is seen for an OH stretching band that would indicate the formation of hydroxyl tautomers within base pairs. Similarly, the relative intensities of the C=O stretching bands of CG in cyclohexane solution remain constant over this same temperature range, confirming that within the base pair, the tautomeric states of the bases remain essentially unperturbed in the 2-amino/6-keto form of G and the 2-keto/4-amino form of C. The spectra of O⁶-MeG aid in the band assignments, since this molecule is frozen in an equivalent of the 2-amino/6-hydroxyl tautomer, but without the OH group and its associated stretching band. We conclude that the probability of tautomerism does not appear to be sufficient to explain the different rotation dynamics for the two amino bonds of the CG base pair. Rather it is argued that mutual polarization within the base pair, which would increase the bond order of the amino bond of C within the base pair, can explain the results without the formation of unconventional tautomers.     

Studies on molecular structure and tautomerism of a vitamin B<Subscript>6</Subscript> analog with density functional theory

This work presents a computational study on the molecular structure and tautomeric equilibria of a novel Schiff base L derived from pyridoxal (PL) and o-phenylenediamine by using the density functional method B3LYP with basis sets 6-31 G(d,p), 6-31++G(d,p), 6-311 G(d,p) and 6-311++G(d,p). The optimized geometrical parameters obtained by B3LYP/6-31 G(d,p) method showed the best agreement with the experimental values. Tautomeric stability study of L inferred that the enolimine form is more stable than its ketoenamine form in both gas phase and solution. However, protonation of the pyridoxal nitrogen atom (LH) have accelerated the formation of ketoenamine form, and therefore, both ketoenamine and enolimine forms could be present in acidic media.     

Structure-activity relationships of alkaloids from mesquite (<Emphasis Type="Italic">Prosopis juliflora</Emphasis> (Sw.) DC.)

The structure–activity relationships of alkaloids (1–5) from mesquite were subjected to assessment of growth inhibition against the shoot and root growth of monocotyledonous plants, barnyard grass, rice and timothy, and dicotyledonous ones, amaranth, lettuce and cress. All alkaloids tested generally showed growth inhibitory against both monocotyledonous and dicotyledonous plants. Furthermore, these alkaloids exhibited higher activity against the growth of root than that of shoot of all plant species used, except that juliprosopine (5) showed higher activity against the shoot growth than the root growth of rice seedling. Among these alkaloids, the highest active compound appeared to be juliprosine (4), followed by a (1:1) mixture of 3-oxo- and 3-oxo-juliprosine (3a and 3b), and juliprosopine (5). The activity of juliprosine (4) containing 2-methyl piperidine bearing hydroxyl groups at C-3 and C-3 was higher than that of 3-oxo- and 3-oxo-juliprosine (3a and 3b) containing 3-oxo- and 3-oxo-2-methylpiperidine. Compound 3 and 4 containing dihydroindolizinium ring showed higher activity than compound 5 containing tetrahydroindolizine ring, whereas compound 1 containing tetrahydroindolizinone ring showed weaker activity. The activity of secojuliprosopinal (2) without indolizine ring was very weak. It was thus clarified that the active sites in the chemical structure of alkaloids from mesquite are the functional group at C-3 and C-3 of piperidine and indolizine skeleton.     

Influence of Acetyl and Bromine Substitutions on Stacking. Crystal and Molecular Structures of 8-Bromo 2′,3′,5′-Triacetyl Adenosine and 8-Bromo 2′,3′,5′-Triacetyl Guanosine

Abstract

The three dimensional structures of 8-bromo 2′,3′,5′-triacetyl adenosine (8-Br Tri A) and 8-bromo 2′,3′,5′-triacetyl guanosine (8-Br Tri G) have been determined by single crystal X-ray diffraction methods to study the combined effect of bromine and acetyl substitutions on molecular conformation and interactions. The ribose puckers differ from those found in unbrominated Tri A and Tri G and unacetylated 8-Br A and 8-Br G analogues. The adenine bases in 8-Br Tri A form A.A.A base triplets using both Watson-Crick and Hoogsteen sites. Br atoms are not involved in stacking unlike most halogenated structures. The ‘scorpion tail’ positioning of acetyl over base in 8-Br Tri G is different from Tri G and is an interesting consequence of bromine substitution.     

NMR based structural studies decipher stacking of the alkaloid coralyne to terminal guanines at two different sites in parallel G-quadruplex DNA, [d(TTGGGGT)]4 and [d(TTAGGGT)]4

BackgroundTelomere elongation by telomerase gets inhibited by G-quadruplex DNA found in its guanine rich region. Stabilization of G-quadruplex DNA upon ligand binding has evolved as a promising strategy to target cancer cells in which telomerase is over expressed.MethodsInteraction of anti-leukemic alkaloid, coralyne, to tetrameric parallel [d(TTGGGGT)]₄ (Ttel7), [d(TTAGGGT)]₄ (Htel7) and monomeric anti-parallel [dGGGG(TTGGGG)₃] (Ttel22) G-quadruplex DNA has been studied using Circular Dichroism (CD) spectroscopy. Titrations of coralyne with Ttel7 and Htel7 were monitored by ¹H and ³¹P NMR spectroscopy. Solution structure of coralyne-Ttel7 complex was obtained by restrained Molecular Dynamics (rMD) simulations using distance restraints from 2D NOESY spectra. Thermal stabilization of DNA was determined by absorption, CD and ¹H NMR.Results and conclusionsBinding of coralyne to Ttel7/Htel7 induces negative CD band at 315/300 nm. A significant upfield shift in all GNH, downfield shift in T2/T7 base protons and upfield shift (1.8 ppm) in coralyne protons indicates stacking interactions. ³¹P chemical shifts and NOE contacts of G3, G6, T2, T7 protons with methoxy protons reveal proximity of coralyne to T2pG3 and G6pT7 sites. Solution structure reveals stacking of coralyne at G6pT7 and T2pG3 steps with two methoxy groups of coralyne located in the grooves along with formation of a hydrogen bond. Binding stabilizes Ttel7/Htel7 by ~ 25–35 °C in 2:1 coralyne-Ttel7/Htel7 complex.General significanceThe present study is the first report on solution structure of coralyne-Ttel7 complex showing stacking of coralyne with terminal guanine tetrads leading to significant thermal stabilization, which may be responsible for telomerase inhibition.     

Mutation Induced by Deoxyxanthosine in Codon 12 of A Synthetic c-Ha-ras Gene

Abstract

To evaluate the base-pairing properties and mutagenicity of deoxyxanthosine in DNA, the modified base was incorporated into a synthetic c-Ha-ras gene and a DNA transfection experiment was done. The ras gene containing deoxyxanthosine showed very high focus-forming activity. Analysis of the genes from transformants showed almost exclusively a transition of G to A. These results indicate that dTMP was preferentially incorporated at the site opposite to deoxyxanthosine, and deoxyxanthosine can induce G to A transitions in mammalian cells.     

Search for structures,potential energy surfaces,and stabilities of planar B<Subscript>n</Subscript>P(n = 1 ∼ 7)

We have systematically explored and investigated the geometrical structures, stability, growth pattern, bonding character, and potential energy surface (PES) of the possible isomers of each cluster for planar B_nP (n = 1 ∼ 7) at the CCSD(T)/6-311+;G(d)//B3LYP/6-311+G(d) level. A large number of planar structures for the possible isomers of B_nP (n = 1 ∼ 7) and transition states are located. Isomers 1a ∼ 7a of B_nP are the lowest-energy structures and 2a, 4a, as well as 6a are more stable than their neighbors. For the lowest-energy structures (1a ∼ 7a) of B_nP, P atom lies at the apex and tends to form two B-P bonds with boron atoms. They exhibit planar zigzag growth feature or approximately spherical-like growth pattern. Results from molecular orbital analysis demonstrate that the formation of the delocalized π MOs and the σ-radial and σ-tangential MOs plays a critical role in stabilizing the structures of lowest-energy isomers (2a ∼ 7a) of B_nP. Importantly, isomers 3a, 3c, 3d, 4a, 4b, 5b, and 5c of B_nP are stable both thermodynamically and kinetically at the CCSD(T)/6-311+G(d)// B3LYP/6-311+G(d) level and detectable in laboratory, which is valuable for further experimental studies of B_nP.     

Dietary Vitamin D3 Restriction Exacerbates Disease Pathophysiology in the Spinal Cord of the G93A Mouse Model of Amyotrophic Lateral Sclerosis

BackgroundDietary vitamin D₃ (D₃) restriction reduces paw grip endurance and motor performance in G93A mice, and increases inflammation and apoptosis in the quadríceps of females. ALS, a neuromuscular disease, causes progressive degeneration of motor neurons in the brain and spinal cord.ObjectiveWe analyzed the spinal cords of G93A mice following dietary D₃ restriction at 2.5% the adequate intake (AI) for oxidative damage (4-HNE, 3-NY), antioxidant enzymes (SOD2, catalase, GPx1), inflammation (TNF-α, IL-6, IL-10), apoptosis (bax/bcl-2 ratio, cleaved/pro-caspase 3 ratio), neurotrophic factor (GDNF) and neuron count (ChAT, SMI-36/SMI-32 ratio).MethodsBeginning at age 25 d, 42 G93A mice were provided food ad libitum with either adequate (AI;1 IU D₃/g feed; 12 M, 11 F) or deficient (DEF; 0.025 IU D₃/g feed; 10 M, 9 F) D₃. At age 113 d, the spinal cords were analyzed for protein content. Differences were considered significant at P ≤ 0.10, since this was a pilot study.ResultsDEF mice had 16% higher 4-HNE (P = 0.056), 12% higher GPx1 (P = 0.057) and 23% higher Bax/Bcl2 ratio (P = 0.076) vs. AI. DEF females had 29% higher GPx1 (P = 0.001) and 22% higher IL-6 (P = 0.077) vs. AI females. DEF males had 23% higher 4-HNE (P = 0.066) and 18% lower SOD2 (P = 0.034) vs. AI males. DEF males had 27% lower SOD2 (P = 0.004), 17% lower GPx1 (P = 0.070), 29% lower IL-6 (P = 0.023) and 22% lower ChAT (P = 0.082) vs. DEF females.ConclusionD₃ deficiency exacerbates disease pathophysiology in the spinal cord of G93A mice, the exact mechanisms are sex-specific. This is in accord with our previous results in the quadriceps, as well as functional and disease outcomes.     

DNA Intercalation and Photoinduced Cleavage by 4-Nitro(N-Hexylamine)1,8-Naphthalimide

Abstract

A novel intercalator, 4-nitro(N-hexylamine)1,8-naphthalimide, was synthesised and its DNA binding and photoinduced DNA cleavage properties were studied. The DNA unwinding results show that it binds through intercalation. Absorption and fluorescence spectroscopy reveal the preference for A/T base pairs as compared to G/C base pairs for the binding. The intercalator produces photoinduced single strand scissions in double helical DNA.