Preparation of iron oxide silica particles for Zika viral RNA extraction.

In this work, a robust synthetic pathway for magnetic core preparation and silica surface coating of magnetic microparticles is presented. Silica-coated magnetic particles are widely used to extract DNA and RNA from various biological samples. We present a novel route for the synthesis of iron oxide silica particles (Fe3O4@Silica) and demonstrate their performance for extracting ZIKA viral RNA from serum. The iron (II, III) oxide (Fe3O4), magnetite core is first prepared by ammonia neutralization of ferrous and ferric chloride aqueous solution under argon, followed by the addition of citrate salt to stabilize the surface of the resultant magnetic nanospheres. After this one-pot, two-step synthesis, the magnetic nanospheres are consumed during silica coating by hydrolysis of tetraethoxysilane (TEOS) under alkaline condition. The final product is a sphere-like magnetic aggregate with a size range of 1-2 micron. By simply suspending the magnetic aggregates in guanidinium chloride solution, the silica surface can be prepared for RNA binding. The RNA extraction efficiency was evaluated by extracting ZIKA viral RNA from serum followed by a PCR-based assay. The data indicate excellent recovery of target RNA and removal of PCR inhibitors. This manufacturing procedure for the silica coated microparticles provides a low-cost, effective and ready for scale-up method whose performance is equivalent to commercial alternatives such as magnetic silica surface particles for DNA and RNA sample preparations. The cost of the clinical assays could be largely decreased due to the 100 fold reduction in cost by replacing the commercially available magnetic particles with the developed material for RNA extraction.

Structure and replication of yDNA: a novel genetic set widened by benzo-homologation.

In a functioning genetic system, the information-encoding molecule must form a regular self-complementary complex (for example, the base-paired double helix of DNA) and it must be able to encode information and pass it on to new generations. Here we study a benzo-widened DNA-like molecule (yDNA) as a candidate for an alternative genetic set, and we explicitly test these two structural and functional requirements. The solution structure of a 10 bp yDNA duplex is measured by using 2D-NMR methods for a simple sequence composed of T-yA/yA-T pairs. The data confirm an antiparallel, right-handed, hydrogen-bonded helix resembling B-DNA but with a wider diameter and enlarged base-pair size. In addition to this, the abilities of two different polymerase enzymes (Klenow fragment of DNA pol I (Kf) and the repair enzyme Dpo4) to synthesize and extend the yDNA pairs T-yA, A-yT, and G-yC are measured by steady-state kinetics studies. Not surprisingly, insertion of complementary bases opposite yDNA bases is inefficient due to the larger base-pair size. We find that correct pairing occurs in several cases by both enzymes, but that common and relatively efficient mispairing involving T-yT and T-yC pairs interferes with fully correct formation and extension of pairs by these polymerases. Interestingly, the data show that extension of the large pairs is considerably more efficient with the flexible repair enzyme (Dpo4) than with the more rigid Kf enzyme. The results shed light on the properties of yDNA as a candidate for an alternative genetic information-encoding molecule and as a tool for application in basic science and biomedicine.

Polymerase amplification, cloning, and gene expression of benzo-homologous "yDNA" base pairs.

A widened DNA base-pair architecture is studied in an effort to explore the possibility of whether new genetic system designs might possess some of the functions of natural DNA. In the "yDNA" system, pairs are homologated by addition of a benzene ring, which yields (in the present study) benzopyrimidines that are correctly paired with purines. Here we report initial tests of ability of the benzopyrimidines yT and yC to store and transfer biochemical and biological information in vitro and in bacterial cells. In vitro primer extension studies with two polymerases showed that the enzymes could insert the correct nucleotides opposite these yDNA bases, but with low selectivity. PCR amplifications with a thermostable polymerase resulted in correct pairings in 15-20 % of the cases, and more successfully when yT or yC were situated within the primers. Segments of DNA containing one or two yDNA bases were then ligated into a plasmid and tested for their ability to successfully lead the expression of an active protein in vivo. Although active at only a fraction of the activity of fully natural DNA, the unnatural bases encoded the correct codon bases in the majority of cases when singly substituted, and yielded functioning green fluorescent protein. Although the activities with native polymerases are modest with these large base pairs, this is the first example of encoding protein in vivo by an unnatural DNA base pair architecture.

Synthesis and properties of size-expanded DNAs: toward designed, functional genetic systems.

We describe the design, synthesis, and properties of DNA-like molecules in which the base pairs are expanded by benzo homologation. The resulting size-expanded genetic helices are called xDNA ("expanded DNA") and yDNA ("wide DNA"). The large component bases are fluorescent, and they display high stacking affinity. When singly substituted into natural DNA, they are destabilizing because the benzo-expanded base pair size is too large for the natural helix. However, when all base pairs are expanded, xDNA and yDNA form highly stable, sequence-selective double helices. The size-expanded DNAs are candidates for components of new, functioning genetic systems. In addition, the fluorescence of expanded DNA bases makes them potentially useful in probing nucleic acids.

Exploring the limits of DNA size: naphtho-homologated DNA bases and pairs.

A new design for DNA bases and base pairs is described in which the pyrimidine bases are widened by naphtho-homologation. Two naphtho-homologated deoxyribosides, dyyT (1) and dyyC (2), were synthesized and could be incorporated into oligonucleotides as suitably protected phosphoramidite derivatives. The deoxyribosides were found to be fluorescent, with emission maxima at 446 and 433 nm, respectively. Studies with single substitutions of 1 and 2 in the natural DNA context revealed exceptionally strong base stacking propensity for both. Sequences containing multiple substitutions of 1 and 2 paired opposite adenine and guanine were subsequently mixed and studied by several analytical methods. Data from UV mixing experiments, FRET measurements, fluorescence quenching experiments, and hybridizations on beads suggest that complementary "doublewide DNA" (yyDNA) strands may self-assemble into helical complexes with 1:1 stoichiometry. Data from thermal denaturation plots and CD spectra were less conclusive. Control experiments in one sequence context gave evidence that yyDNA helices, if formed, are preferentially antiparallel and are sequence selective. Hypothesized base pairing schemes are analogous to Watson-Crick pairing, but with glycosidic C1'-C1' distances widened by over 45%, to ca. 15.2 A. The possible self-assembly of the double-wide DNA helix establishes a new limit for the size of information-encoding, DNA-like molecules, and the fluorescence of yyDNA bases suggests uses as reporters in monomeric and oligomeric forms.

A new four-base genetic helix, yDNA, composed of widened benzopyrimidine-purine pairs.

We describe the properties of stable DNA-like self-assembled helices composed entirely of base pairs involving two new size-expanded pyrimidines. We term this new helix geometry "yDNA" (an abbreviation of "wide DNA"). The new pyrimidine analogues, yT and yC, are increased in size by benzo-homologation and have a geometry that is distinct from previous size-expanded pyrimidines. The yT and yC deoxyribosides were incorporated into oligodeoxynucleotides designed to form four pairs: yT-A, A-yT, yC-G, and G-yC. Helices were characterized by thermal denaturation, mixing data, and circular dichroism spectra. Results showed that highly stable double-stranded helices were formed in several sequence contexts. The data further showed that yT and yC could be segregated onto one strand and used to bind to natural strands of DNA with high sequence selectivity. The combination of yC, yT, G, and A make up a new selective, self-assembling four-base genetic pairing system that functions in many respects like natural DNA, but which is structurally distinct. The results establish that multiple variants of size-expanded DNA-like helices are feasible and suggest the possibility of a future eight-base genetic system based on the yDNA geometry. Finally, the high binding selectivity, affinity, and fluorescence of yDNA strands may yield useful applications in detection of nucleic acid sequences.

Novel benzopyrimidines as widened analogues of DNA bases.

We report on the synthesis, stacking, and pairing properties of a new structural class of size-expanded pyrimidine nucleosides, abbreviated dyT and dyC. Their bases are benzo-homologated variants of thymine and cytosine and have a design that is distinct from a previously described class of size-expanded (xDNA) pyrimidines, with a different vector of expansion relative to the sugar. We term this new base geometry "yDNA" (a mnemonic for "wide DNA"). Both C-glycosides were prepared using Pd-mediated coupling of iodinated base derivatives with a deoxyribose precursor. As free deoxynucleosides, both dyT and dyC displayed robust fluorescence, with emission maxima at 375 and 390 nm, respectively. Both widened pyrimidines could be incorporated readily as protected phosphoramidite derivatives into synthetic oligonucleotides. Experiments in "dangling end" DNA contexts revealed that both yT and yC stack more favorably than their natural counterparts. When opposite natural bases in the context of Watson-Crick DNA were paired, the yT nucleotide formed a pair with A that was equally stable as a T-A pair, despite the mismatch in size with the neighboring natural pairs. The yC nucleotide (paired opposite G) was destabilizing by a small amount in the same context. Despite the large size of the pairs, both yT and yC were selective for their Watson-Crick complementary partners A and G, respectively. The pairing properties and fluorescence of yDNA nucleotides may lead to useful applications in the study of steric effects in DNA-protein interactions. In addition, the compounds may serve as building blocks for a large-sized artificial genetic system.

Acid-promoted DNA-cleaving activities and total synthesis of varacin C.

The first total synthesis of the antibiotic varacin C has been accomplished. In addition, it has been demonstrated that this antibiotic exhibits potent antitumor activity and is capable of causing efficient DNA cleavage under acidic conditions.

Benzotrithiole 2-oxide: a new family of thiol-activated DNA-cleaving functionalities.

It was demonstrated in our studies that benzotrithiole 2-oxide was capable of causing efficient DNA cleavage in the presence of 2-mercaptoethanol or glutathione and exhibited potent cytotoxic properties against certain cancer cell lines.

Acid-accelerated DNA-cleaving activities of antitumor antibiotic varacin.

It is demonstrated in this report that the authentic molecular structure of antibiotic varacin is capable of causing DNA-cleavage with high efficiency in the presence of thiols. In addition, it is found that the DNA-cleaving activity by varacin is apparently promoted by its acidic environments.

G-quadruplex as a new class of structural entities for directing the formation of circular oligodeoxyribonucleotides.

It has been demonstrated for the first time that G-quadruplex is capable of acting as a template for directing the sequence-specific formation of certain circular oligodeoxyribonucleotides with high efficiency.