Solution Structure,Mechanism of Replication,and Optimization of an Unnatural Base Pair

As part of an ongoing effort to expand the genetic alphabet for in vitro and eventual in vivo applications, we have synthesized a wide variety of predominantly hydrophobic unnatural base pairs and evaluated their replication in DNA. Collectively, the results have led us to propose that these base pairs, which lack stabilizing edge‐on interactions, are replicated by means of a unique intercalative mechanism. Here, we report the synthesis and characterization of three novel derivatives of the nucleotide analogue d MMO2 , which forms an unnatural base pair with the nucleotide analogue d 5SICS . Replacing the para‐methyl substituent of d MMO2 with an annulated furan ring (yielding d FMO ) has a dramatically negative effect on replication, while replacing it with a methoxy (d DMO ) or with a thiomethyl group (d TMO ) improves replication in both steady‐state assays and during PCR amplification. Thus, d TMO –d 5SICS , and especially d DMO –d 5SICS , represent significant progress toward the expansion of the genetic alphabet. To elucidate the structure–activity relationships governing unnatural base pair replication, we determined the solution structure of duplex DNA containing the parental d MMO2 –d 5SICS pair, and also used this structure to generate models of the derivative base pairs. The results strongly support the intercalative mechanism of replication, reveal a surprisingly high level of specificity that may be achieved by optimizing packing interactions, and should prove invaluable for the further optimization of the unnatural base pair.     

碱基对在DNA双螺旋链上分离的自由能计算

DNA是大部分生物包括病毒的基因载体。DNA双螺旋链通过A＝T和G≡C两种碱基对编码实现对遗传信息的存储。碱基对中的相互作用对DNA双螺旋链的稳定性起到重要作用,直接关系到基因的复制和转录。当前研究中,我们构建了四组不同结构的DNA双螺旋链,进行了总共4.3 μs的分子动力学模拟。通过伞形取样技术计算了DNA双螺旋链中碱基对分离的自由能曲线,并从分子尺度细节和相互作用能对自由能曲线进行解析。在碱基对G≡C的自由能曲线(PMF-PGC)上观察到三个峰,通过监测氢键数目的变化发现分别对应于G≡C三个氢键的断裂;而在A＝T的自由能曲线(PMF-PAT)上只出现一个峰,说明A＝T的两个氢键在分离过程中几乎同时断裂。PMF-PGC的总能垒比PMF-PAT高,主要是因为G≡C比A＝T多一个氢键,更稳定。两条曲线的后段自由能仍然升高,而此时碱基对的氢键已断裂,这是DNA链骨架刚性所导致。我们还研究了碱基对稳定性受相邻碱基对的影响,发现邻近G≡C碱基对会增强A＝T的稳定性, C≡G会削弱A＝T的稳定性, T＝A对A＝T的影响较小。     

B型DNA中鸟嘌呤-胞嘧啶碱基对内双质子转移反应

采用ONIOM(M06-2X/6-31G^*:PM3)方法研究了单个鸟嘌呤-胞嘧啶(GC)碱基对和含GC碱基对的四种排序的DNA三聚体(dATGCAT, dGCGCGC, dTAGCTA, dCGGCCG)的双质子转移反应. 通过分析其双质子转移方式、质子转移过程中各结构的能量和氢键变化, 总结出环境因素对GC碱基对双质子转移机理的影响. 气相中, dCGGCCG三聚体中发生分步双质子转移, 其它四种模型中均发生协同双质子转移. 分析发现质子转移方式受上下相邻碱基对的静电相互作用和质子接受位的质子亲和势影响, dATGCAT和dGCGCGC排序有助于质子H4a转移, 而dTAGCTA和dCGGCCG排序有助于质子H1转移, 胞嘧啶的N3位较高的质子亲和势有助于质子H1转移. 水溶剂中, 上下相邻碱基对的静电相互作用被减弱, 水溶剂稳定了分步转移过程中的单质子转移产物, 因此分步转移机理占据优势, 五种模型中均出现分步双质子转移, 在此过程中能量变化趋势相似. 溶剂效应有利于单质子转移, 却增加了双质子转移反应的反应能.     

Cellular Target of a Rhodium Metalloinsertor is the DNA Base Pair Mismatch

Defects in DNA mismatch repair (MMR) are commonly found in various cancers, especially in colorectal cancers. Despite the high prevalence of MMR-deficient cancers, mismatch-targeted therapeutics are limited and diagnostic tools are indirect. Here, we examine the cytotoxic properties of a rhodium metalloinsertor, [Rh(phen)(chrysi)(PPO)]²⁺ ( RhPPO ) in 27 diverse colorectal cancer cell lines. Despite the low frequency of genomic mismatches and the non-covalent nature of the RhPPO -DNA lesion, RhPPO is on average five times more potent than cisplatin. Importantly, the biological target and profile for RhPPO differs from that of cisplatin. A fluorescent metalloinsertor, RhCy3 , was used to demonstrate that the cellular target of RhPPO is the DNA mismatch. RhCy3 represents a direct probe for MMR-deficiency and correlates directly with the cytotoxicity of RhPPO across different cell lines. Overall, our studies clearly indicate that RhPPO and RhCy3 are promising anticancer and diagnostic probes for MMR-deficient cancers, respectively.     

Tailored Tolane-Perfluorotolane Assembly as Supramolecular Base Pair Replacement in DNA

Arene-fluoroarene interactions offer outstanding possibilities for engineering of supramolecular systems, including nucleic acids. Here, we implement the tolane-perfluorotolane interaction as base pair replacement in DNA. Tolane (THH) and perfluorotolane (TFF) moieties were connected to acyclic backbone units, comprising glycol nucleic acid (GNA) or butyl nucleic acid (BuNA) building blocks, that were incorporated via phosphoramidite chemistry at opposite positions in a DNA duplex. Thermodynamic analyses by UV thermal melting revealed a compelling stabilization by THH/TFF heteropairs only when connected to the BuNA backbone, but not with the shorter GNA linker. Detailed NMR studies confirmed the preference of the BuNA backbone for enhanced polar π-stacking. This work defines how orthogonal supramolecular interactions can be tailored by small constitutional changes in the DNA backbone, and it inspires future studies of arene-fluoroarene-programmed assembly of DNA.     

Development of a Metal‐Ion‐Mediated Base Pair for Electron Transfer in DNA

A new C‐nucleoside structurally based on the hydroxyquinoline ligand was synthesized that is able to form stable pairs in DNA in both the absence and the presence of metal ions. The interactions between the metal centers in adjacent Cu^II‐mediated base pairs in DNA were probed by electron paramagnetic resonance (EPR) spectroscopy. The metal–metal distance falls into the range of previously reported values. Fluorescence studies with a donor–DNA–acceptor system indicate that photoinduced charge‐transfer processes across these metal‐ion‐mediated base pairs in DNA occur more efficiently than over natural base pairs.     

Structure Determination of an AgI‐Mediated Cytosine–Cytosine Base Pair within DNA Duplex in Solution with 1H/15N/109Ag NMR Spectroscopy

The structure of an Ag^I‐mediated cytosine–cytosine base pair, C–Ag^I–C, was determined with NMR spectroscopy in solution. The observation of 1‐bond ¹⁵N‐¹⁰⁹Ag J‐coupling (¹J(¹⁵N,¹⁰⁹Ag): 83 and 84 Hz) recorded within the C–Ag^I–C base pair evidenced the N3–Ag^I–N3 linkage in C–Ag^I–C. The triplet resonances of the N4 atoms in C–Ag^I–C demonstrated that each exocyclic N4 atom exists as an amino group (?NH₂), and any isomerization and/or N4–Ag^I bonding can be excluded. The 3D structure of Ag^I–DNA complex determined with NOEs was classified as a B‐form conformation with a notable propeller twist of C–Ag^I–C (?18.3±3.0°). The ¹⁰⁹Ag NMR chemical shift of C‐Ag^I‐C was recorded for cytidine/Ag^I complex (δ(¹⁰⁹Ag): 442 ppm) to completed full NMR characterization of the metal linkage. The structural interpretation of NMR data with quantum mechanical calculations corroborated the structure of the C–Ag^I–C base pair.     

Infrared Spectroscopic Observation of a G–C+ Hoogsteen Base Pair in the DNA:TATA‐Box Binding Protein Complex Under Solution Conditions

Hoogsteen DNA base pairs (bps) are an alternative base pairing to canonical Watson–Crick bps and are thought to play important biochemical roles. Hoogsteen bps have been reported in a handful of X‐ray structures of protein–DNA complexes. However, there are several examples of Hoogsteen bps in crystal structures that form Watson–Crick bps when examined under solution conditions. Furthermore, Hoogsteen bps can sometimes be difficult to resolve in DNA:protein complexes by X‐ray crystallography due to ambiguous electron density and by solution‐state NMR spectroscopy due to size limitations. Here, using infrared spectroscopy, we report the first direct solution‐state observation of a Hoogsteen (G–C⁺) bp in a DNA:protein complex under solution conditions with specific application to DNA‐bound TATA‐box binding protein. These results support a previous assignment of a G–C⁺ Hoogsteen bp in the complex, and indicate that Hoogsteen bps do indeed exist under solution conditions in DNA:protein complexes.     

A Dinuclear Mercury(II)‐Mediated Base Pair in DNA

The first dinuclear metal‐mediated base pair containing divalent metal ions has been prepared. A combination of the neutral bis(monodentate) purine derivative 1,N⁶‐ethenoadenine (ϵA), which preferentially binds two metal ions with a parallel alignment of the N−M bonds, and the canonical nucleobase thymine (T), which readily deprotonates in the presence of Hg^II and thereby partially compensates the charge accumulation due to the two closely spaced divalent metal ions, yields the dinuclear T‐Hg^II₂‐ϵA base pair. This metal‐mediated base pair stabilizes the DNA oligonucleotide duplex as shown by an increase of 8 °C in its melting temperature. Formation of the base pair was demonstrated by temperature‐dependent UV spectroscopy as well as by titration experiments monitored by UV and CD spectroscopy.     

Enzymatic Incorporation of a Coumarin–Guanine Base Pair

Previous expansions beyond nature's preferred base‐pairing interactions have utilized either nonpolar shape‐fitting interactions or classical hydrogen bonding. Reported here is a hybrid of these systems. By replacing a single N?H with C?H at a Watson–Crick interface, the design space for new drug candidates and fluorescent nucleobase analogues is dramatically expanded, as demonstrated here by the new, highly fluorescent deoxycytidine mimic 3‐glycosyl‐5‐fluoro‐7‐methoxy‐coumarin‐2′‐deoxyribose (d C ^C ). dGTP is selectively incorporated across from a template d C ^C during enzymatic DNA synthesis. Likewise, d C ^C is selectively incorporated across from a template guanine when d C ^C is provided as the triphosphate d C ^C TP . DNA polymerase I (Klenow fragment) exhibited about a 10‐fold higher affinity for d C ^C TP than dCTP, allowing selective incorporation of d C ^C in direct competition experiments. These results demonstrate that a single C?H can replace N?H at a Watson–Crick‐type interface with preservation of functional selectivity and enhanced activity.     

Synthesis and Properties of Nonpolar DNA (Arylalkyl)phosphonates

The eight (arylalkyl)‐modified phosphoramidites (=(arylalkyl)phosphonamidites) 1 – 8 (Fig. 2) were synthesized (Schemes 1–3) and incorporated at different positions into 2′‐deoxyoligonucleotides. The [P(R)]‐ and [P(S)]‐diastereoisomers of the hexanucleotides 32 – 39 (Table 1) and of the dodecanucleotides 41 – 45 (Table 2) obtained were separated by means of reversed‐phase HPLC. UV, CD, and fluorescence spectroscopy were used to investigate the thermal stability (T_m) and the structural changes of their DNA duplexes with 5′‐d(CGCGCG)‐3′ and 5′‐d(ATGATTGACCTG)‐3′, respectively. The T_m values significantly depend on the place of modification (Table 2). A dangling‐end effect is observed when the [3‐(anthracen‐9‐yl)propyl]‐modified 8 is attached at the 5′‐terminus (see duplex with 45c ). In the case of the incorporation of aromatic moieties tethered via a methylene linker to the P‐atom (benzyl‐ and (naphthalen‐1‐ylmethyl)‐modified 1 and 6 , resp.), the duplexes with the [P(R)]‐oligonucleotides are more stable than those with the [P(S)]‐isomers, whereas in the case of longer alkyl chains at the P‐atom (see 2 – 5 ), the T_m values show the reverse tendency. The observed T_m differences are assigned to changes in base stacking (Figs. 6 and 7).     

Influence of the Absolute Configuration of NPE‐Caged Cytosine on DNA Single Base Pair Stability

Photolabile protecting groups are a versatile tool to trigger reactions by light irradiation. In this study, we have investigated the influence of the absolute configuration of the 1‐(2‐nitrophenyl)ethyl (NPE) cage group on a 15‐base‐pair duplex DNA. Using UV melting, we determined the global stability of the unmodified and the selectively (S)‐ and (R)‐NPE‐modified DNA sequences, respectively. We observe a differently destabilizing effect for the two NPE stereoisomers on the global stability. Analysis of the temperature dependence of imino proton exchange rates measured by NMR spectroscopy reveals that this effect can be attributed to decreased base pair stabilities of the caged and the 3′‐neighbouring base pair, respectively. Furthermore, our NMR based structural models of the modified duplexes provide a structural basis for the distinct effect of the (S)‐ and the (R)‐NPE group.     

生物大分子的单分子力谱:在水溶液与非极性溶剂中的对照研究

生物大分子是构成生命的物质基础,在其天然环境——水溶液中一般以精确的超分子结构存在.迄今,人们已经合成了种类繁多的水溶性高分子.然而,鲜有合成高分子能够在水溶液中完成精确的超分子组装.与合成高分子相比,生物大分子是特殊而神奇的.为了研究生物大分子与水的相互作用,近年来作者以单分子力谱为主要的实验方法,开展了生物大分子在水溶液与非极性溶剂中的对照研究.研究表明,在非极性溶剂中,生物大分子的超分子结构失稳,转变为无超分子结构的状态.水是一个重要的开关,调控着生物大分子的超分子结构和功能.作者据此提出了生物大分子的水环境适应性概念和早期化学进化过程中水环境筛选生物大分子的假说,并认为水环境适应性是生物大分子和合成水溶性高分子的分水岭.对水和生物大分子的深入研究,将有望破解生命的更多奥秘.     

Merocyanine 540 Solubilized as an Ion Pair with Cationic Surfactant in Nonpolar Solvents: Spectral and Photochemical Properties

Merocyanine 540 (MC) is an anionic dye that is used to photopurge the bone marrow of leukemia cells. Under these conditions it is localized mostly in cell membranes, which may affect its photochemical reactivity. We investigated the photochemistry of MC dissolved as a hydrophobic ion pair with a hexadecyltrioctadecylammonium cation in cyclohexane, trimethylpentane and toluene as well as in propylene carbonate, CH3CN, C2H5OH and D2O. In organic solvents, the absorption and fluorescence spectra of MC were strongly red-shifted compared with aqueous solutions. The fluorescence was also more intense despite aggregation that occurred in some solvents. Aggregation strongly affects the spectral and photochemical properties of MC, especially in aliphatic hydrocarbons in which distinctive H-type aggregates are formed. Hydrophobic MC is a moderate photosensitizer of singlet molecular oxygen (1O2). The following values for 1O2 quantum yields were calculated based on 1O2 phosphorescence relative to 1O2 generation by Rose Bengal: approximately 0.12 in trimethylpenthane, approximately 0.13 in cyclohexane, 0.045 in EtOH, 0.039 in toluene, 0.007 in CH3CN and approximately 3 x 10(-4) in D2O. The H-aggregates of MC in cyclohexane and trimethylpentane are better 1O2 producers than monomeric MC. The above 1O2 quantum yields are corrected for self-quenching because MC is an efficient 1O2 quencher (17 x 10(7) M-1 s-1 in CH3CN, 6.8 x 10(7) M-1 s-1 in D2O, 5.2 x 10(7) M-1 s-1 in EtOH, and 1.4 x 10(7) M-1 s-1 in toluene). Merocyanine undergoes photodegradation, a solvent-dependent process that proceeds faster when the dye is aggregated. The initial photodegradation rate is much slower in organic solvents than in water, but photodegradation products accumulated during longer irradiation may increase the rate in most solvents. Higher photostability and better photosensitization by MC in hydrophobic nonpolar solvents suggest that the killing of leukemia cells via a photodynamic mechanism may operate mostly in cell membranes. In contrast, any cytotoxic products from photodecomposition may be important in hydrophilic cell compartments. Our data show the spectral and photochemical properties of MC in a pure hydrophobic environment.     

Solution Structure of a Lanthanide-binding DNA Aptamer Determined Using High Quality pseudocontact shift restraints

In this contribution we report the high-resolution NMR structure of a recently identified lanthanide-binding aptamer (LnA). We demonstrate that the rigid lanthanide binding by LnA allows for the measurement of anisotropic paramagnetic NMR restraints which to date remain largely inaccessible for nucleic acids. One type of such restraints - pseudocontact shifts (PCS) induced by four different paramagnetic lanthanides - was extensively used throughout the current structure determination study and the measured PCS turned out to be exceptionally well reproduced by the final aptamer structure. This finding opens the perspective for a broader application of paramagnetic effects in NMR studies of nucleic acids through the transplantation of the binding site found in LnA into other DNA/RNA systems.