Aaron E. Engelhart

DNA and RNA in Anhydrous Media: Duplex, Triplex, and G‐Quadruplex Secondary Structures in a Deep Eutectic Solvent

These eutecticmixtures are attractive alternatives to RTILs, as DESs can beless expensive, more synthetically accessible, nontoxic, andbiodegradable. Herein, we report that nucleic acids can formseveral secondary structures that reversibly denature withheating in a water-free DES. In some cases, the nucleic acidsequences studied exhibited different relative stabilities anddifferent secondary structures in the DES to those in aqueousmedia. The results presented suggest that DESs and RTILscan be used as media for nucleic acid based technologies, andthey have direct implications regarding the perceived neces-sity of water for nucleic acid secondary structure.

Primitive Genetic Polymers

Since the structure of DNA was elucidated more than 50 years ago, Watson-Crick base pairing has been widely speculated to be the likely mode of both information storage and transfer in the earliest genetic polymers. The discovery of catalytic RNA molecules subsequently provided support for the hypothesis that RNA was perhaps even the first polymer of life. However, the de novo synthesis of RNA using only plausible prebiotic chemistry has proven difficult, to say the least. Experimental investigations, made possible by the application of synthetic and physical organic chemistry, have now provided evidence that the nucleobases (A, G, C, and T/U), the trifunctional moiety ([deoxy]ribose), and the linkage chemistry (phosphate esters) of contemporary nucleic acids may be optimally suited for their present roles-a situation that suggests refinement by evolution. Here, we consider studies of variations in these three distinct components of nucleic acids with regard to the question: Is RNA, as is generally acknowledged of DNA, the product of evolution? If so, what chemical and structural features might have been more likely and advantageous for a proto-RNA?

Conformational Variants of Duplex DNA Correlated with Cytosine-rich Chromosomal Fragile Sites

We found that several major chromosomal fragile sites in human lymphomas, including the bcl-2 major breakpoint region, bcl-1 major translocation cluster, and c-Myc exon 1-intron 1 boundary, contain distinctive sequences of consecutive cytosines exhibiting a high degree of reactivity with the structure-specific chemical probe bisulfite. To assess the inherent structural variability of duplex DNA in these regions and to determine the range of structures reactive to bisulfite, we have performed bisulfite probing on genomic DNA in vitro and in situ; on duplex DNA in supercoiled and linearized plasmids; and on oligonucleotide DNA/DNA and DNA/2′-O-methyl RNA duplexes. Bisulfite is significantly more reactive at the frayed ends of DNA duplexes, which is expected given that bisulfite is an established probe of single-stranded DNA. We observed that bisulfite also distinguishes between more subtle sequence/structural differences in duplex DNA. Supercoiled plasmids are more reactive than linear DNA; and sequences containing consecutive cytosines, namely GGGCCC, are more reactive than those with alternating guanine and cytosine, namely GCGCGC. Circular dichroism and x-ray crystallography show that the GGGCCC sequence forms an intermediate B/A structure. Molecular dynamics simulations also predict an intermediate B/A structure for this sequence, and probe calculations suggest greater bisulfite accessibility of cytosine bases in the intermediate B/A structure over canonical B- or A-form DNA. Electrostatic calculations reveal that consecutive cytosine bases create electropositive patches in the major groove, predicting enhanced localization of the bisulfite anion at homo-C tracts over alternating G/C sequences. These characteristics of homo-C tracts in duplex DNA may be associated with DNA-protein interactions in vivo that predispose certain genomic regions to chromosomal fragility.

Intercalation as a means to suppress cyclization and promote polymerization of base-pairing oligonucleotides in a prebiotic world

The RNA world hypothesis proposes that nucleic acids were once responsible for both information storage and chemical catalysis, before the advent of coded protein synthesis. However, it is difficult to imagine how nucleic acid polymers first appeared, as the abiotic chemical formation of long nucleic acid polymers from mononucleotides or short oligonucleotides remains elusive, and barriers to achieving this goal are substantial. One specific obstacle to abiotic nucleic acid polymerization is strand cyclization. Chemically activated short oligonucleotides cyclize efficiently, which severely impairs polymer growth. We show that intercalation, which stabilizes and rigidifies nucleic acid duplexes, almost totally eliminates strand cyclization, allowing for chemical ligation of tetranucleotides into duplex polymers of up to 100 base pairs in length. In contrast, when these reactions are performed in the absence of intercalators, almost exclusively cyclic tetra- and octanucleotides are produced. Intercalator-free polymerization is not observed, even at tetranucleotide concentrations > 10,000-fold greater than those at which intercalators enable polymerization. We also demonstrate that intercalation-mediated polymerization is most favored if the size of the intercalator matches that of the base pair; intercalators that bind to Watson–Crick base pairs promote the polymerization of oligonucleotides that form these base pairs. Additionally, we demonstrate that intercalation-mediated polymerization is possible with an alternative, non-Watson–Crick-paired duplex that selectively binds a complementary intercalator. These results support the hypothesis that intercalators (acting as ‘molecular midwives’) could have facilitated the polymerization of the first nucleic acids and possibly helped select the first base pairs, even if only trace amounts of suitable oligomers were available.

Structural insights into the effects of 2'-5' linkages on the RNA duplex.

Significance The nonenzymatic replication of RNA is thought to have been a critical step in the emergence of simple cellular life from prebiotic chemistry. However, the chemical copying of RNA templates generates product strands that contain 2′-5′ backbone linkages and normal 3′-5′ linkages. Our recent finding that RNAs with such mixed backbones can still fold into functional structures raised the question of how RNA accommodates the presence of 2′-5′ linkages. Here we use X-ray crystallography and molecular dynamics simulations to reveal how 3′-5′–linked RNA duplexes accommodate interspersed 2′-5′ linkages. The diminished thermal and chemical stability of such RNA duplexes reflects local structural changes, but compensatory changes result in a global RNA duplex structure with relatively minor alterations. The mixture of 2′-5′ and 3′-5′ linkages generated during the nonenzymatic replication of RNA has long been regarded as a central problem for the origin of the RNA world. However, we recently observed that both a ribozyme and an RNA aptamer retain considerable functionality in the presence of prebiotically plausible levels of linkage heterogeneity. To better understand the RNA structure and function in the presence of backbone linkage heterogeneity, we obtained high-resolution X-ray crystal structures of a native 10-mer RNA duplex (1.32 Å) and two variants: one containing one 2′-5′ linkage per strand (1.55 Å) and one containing three such linkages per strand (1.20 Å). We found that RNA duplexes adjust their local structures to accommodate the perturbation caused by 2′-5′ linkages, with the flanking nucleotides buffering the disruptive effects of the isomeric linkage and resulting in a minimally altered global structure. Although most 2′-linked sugars were in the expected 2′-endo conformation, some were partially or fully in the 3′-endo conformation, suggesting that the energy difference between these conformations was relatively small. Our structural and molecular dynamic studies also provide insight into the diminished thermal and chemical stability of the duplex state associated with the presence of 2′-5′ linkages. Our results contribute to the view that a low level of 2′-5′ substitution would not have been fatal in an early RNA world and may in contrast have been helpful for both the emergence of nonenzymatic RNA replication and the early evolution of functional RNAs.

Collaboration between primitive cell membranes and soluble catalysts

One widely held model of early life suggests primitive cells consisted of simple RNA-based catalysts within lipid compartments. One possible selective advantage conferred by an encapsulated catalyst is stabilization of the compartment, resulting from catalyst-promoted synthesis of key membrane components. Here we show model protocell vesicles containing an encapsulated enzyme that promotes the synthesis of simple fatty acid derivatives become stabilized to Mg2+, which is required for ribozyme activity and RNA synthesis. Thus, protocells capable of such catalytic transformations would have enjoyed a selective advantage over other protocells in high Mg2+ environments. The synthetic transformation requires both the catalyst and vesicles that solubilize the water-insoluble precursor lipid. We suggest that similar modified lipids could have played a key role in early life, and that primitive lipid membranes and encapsulated catalysts, such as ribozymes, may have acted in conjunction with each other, enabling otherwise-impossible chemical transformations within primordial cells.

Submicromolar, Selective G‐Quadruplex Ligands from One Pot: Thermodynamic and Structural Studies of Human Telomeric DNA Binding by Azacyanines

The discovery of G-quadruplex structures in nucleic acid sequences associated with cancers has created intense interest in G quadruplexes as potential drug targets. These fourstranded structures, with planar G tetrads represent appealing DNA targets (Figure 1A), as they are structurally distinct from the Watson–Crick duplex of most genomic DNA. Small molecules with high affinity and high selectivity for G quadruplexes have even begun to show medicinal promise, although the connection between G-quadruplex binding and in vivo activity might not always be obvious. As one promising example, quarfloxin, an antineoplastic that targets the rRNA–nucleolin complex, is presently in phase II clinical trials. Most investigators who seek new ligands for G-quadruplex DNA have followed two common strategies. First, they have focused on heterocycles with a relatively large and planar surface area, which maximizes stacking with the about 1 nm surface of a G tetrad (e.g. , TmPyP4, Figure 1C). Second, many have used multiple charges to increase electrostatic interactions with the high-charge density G quadruplex (e.g. , BRACO-19, Figure 1D) and to enhance the solubility of potential ligands (often necessary for ligands with large hydrophobic surfaces, e.g. , TmPyP4). While these strategies have resulted in several high affinity ligands for G quadruplexes, most ligands do not exhibit high selectivity over duplex DNA. Recently, several metallated quadruplex ligands have been reported with substantial selectivity for quadruplex DNA. As the authors have suggested, this could be, in part, due to ACHTUNGTRENNUNGinteractions between the metal ion and the lone pairs of the carbonyl oxygen atoms of the exterior quartets, which are not accessible in duplex DNA. Higher-throughput, more combinatorial approaches have begun to show promise as well. For example, Balasubmaranian and co-workers have recently demonstrated dynamic combinatorial selection of quadruplex ligands by disulfide bond formation with a thiol-containing scaffold and side chains. We have taken a different approach to targeting the G quadruplex. We previously discovered that a planar molecule larger than a typical DNA intercalator can selectively bind purine–purine base pairs. Based upon this discovery, we hypothesized that planar, monocationic molecules that are marginally too large to intercalate a Watson–Crick duplex, such as bispurine analogues, might selectively bind G quadruplexes. We report a new class of selective, submicromolar quadruplex ligands with a facile synthetic route: the azacyanines (Figure 1B). The synthesis of azacyanines was previously reported by Kurth and co-workers ; the route is one-pot and workup is by filtration. The route is general and succeeds for aminobenzimidazoles and aminobenzothiazoles. The synthetic ease makes the class amenable to library preparation for highthroughput screening. We investigated the binding of azacyanines to a G-quadruplex sequence, based on the human telomeric repeat dACHTUNGTRENNUNG(TTA-

Generation of functional RNAs from inactive oligonucleotide complexes by non-enzymatic primer extension.

The earliest genomic RNAs had to be short enough for efficient replication, while simultaneously serving as unfolded templates and effective ribozymes. A partial solution to this paradox may lie in the fact that many functional RNAs can self-assemble from multiple fragments. Therefore, in early evolution, genomic RNA fragments could have been significantly shorter than unimolecular functional RNAs. Here, we show that unstable, nonfunctional complexes assembled from even shorter 3′-truncated oligonucleotides can be stabilized and gain function via non-enzymatic primer extension. Such short RNAs could act as good templates due to their minimal length and complex-forming capacity, while their minimal length would facilitate replication by relatively inefficient polymerization reactions. These RNAs could also assemble into nascent functional RNAs and undergo conversion to catalytically active forms, by the same polymerization chemistry used for replication that generated the original short RNAs. Such phenomena could have substantially relaxed requirements for copying efficiency in early nonenzymatic replication systems.

Nonenzymatic Ligation of DNA with a Reversible Step and a Final Linkage that Can Be Used in PCR

Nonenzymatic DNA ligation chemistries containing a reversible step allow thermodynamic control of product formation, but they are not necessarily compatible with polymerase enzymes. We report a ligation system that uses commercially available reagents, includes a reversible step, and results in a linkage that can function as a template for PCR amplification with accurate sequence transfer.

Construction of a liposome dialyzer for the preparation of high-value, small-volume liposome formulations

The liposome dialyzer is a small-volume equilibrium dialysis device, built from commercially available materials, that is designed for the rapid exchange of small volumes of an extraliposomal reagent pool against a liposome preparation. The dialyzer is prepared by modification of commercially available dialysis cartridges (Slide-A-Lyzer cassettes), and it consists of a reactor with two 300-μl chambers and a 1.56-cm2 dialysis surface area. The dialyzer is prepared in three stages: (i) disassembling the dialysis cartridges to obtain the required parts, (ii) assembling the dialyzer and (iii) sealing the dialyzer with epoxy. Preparation of the dialyzer takes ∼1.5 h, not including overnight epoxy curing. Each round of dialysis takes 1–24 h, depending on the analyte and membrane used. We previously used the dialyzer for small-volume non-enzymatic RNA synthesis reactions inside fatty acid vesicles. In this protocol, we demonstrate other applications, including removal of unencapsulated calcein from vesicles, remote loading and vesicle microscopy.