Michael E. Hogan

Intramolecular G-quartet Motifs Confer Nuclease Resistance to a Potent Anti-HIV Oligonucleotide

We have identified a potentially therapeutic anti-human immunodeficiency virus (HIV)-1 oligonucleotide composed entirely of deoxyguanosines and thymidines (T30177, also known as AR177: 5′-g*tggtgggtgggtggg*t-3′, where asterisk indicates phosphorothioate linkage). In acute assay systems using human T-cells, T30177 and its total phosphodiester homologue T30175 inhibited HIV-1-induced syncytium production by 50% at 0.15 and 0.3 μM, respectively. Under physiological conditions, the sequence and composition of the 17-mer favors the formation of a compact, intramolecularly folded structure dominated by two stacked guanine quartet motifs that are connected by three loops of TGs. The molecule is stabilized by the coordination of a potassium ion between the two stacked quartets. We now show that these guanine quartet-containing oligonucleotides are highly resistant to serum nucleases, with tof 5 h and >4 days for T30175 and T30177, respectively. Both oligonucleotides were internalized efficiently by cells, with intracellular concentrations reaching 5-10-fold above the extracellular levels after 24 h of incubation. In contrast, single-base mutated variants or random sequence control oligonucleotides that could not form the compactly folded structure had markedly reduced half-lives (tfrom 3 to 7 min), low cellular uptake, and no sequence-specific anti-HIV-1 activity. These data suggest that the tertiary structure of an oligonucleotide is a key determinant of its nuclease resistance, cellular uptake kinetics, and biological efficacy.

Human therapeutics based on triple helix technology

The application of triple helix technology to rational drug discovery is rapidly leading to the development of a new class of drugs, initially applicable for the effective and specific treatment of viral infections and, eventually, perhaps, cancer and immunological disorders. Issues such as stability, delivery, specificity, in vitro efficacy and acute toxicity, and the cost of synthesis are already being addressed: preliminary studies are yielding promising results. Further chemical modification of the oligonucleotides will probably be necessary to enhance affinity and efficacy, and comprehensive studies on the pharmacokinetics, toxicity, mutagenicity and in vivo efficacy of these compounds are still required. These, as well as scale-up synthesis and pharmaceutical formulation issues, are the focus of the R&D programs of a number of pharmaceutical laboratories.

Potassium-Induced Loop Conformational Transition of a Potent Anti-HIV Oligonucleotide

Spectroscopic, thermal denaturation and kinetic studies have revealed that DNA oligonucleotides 5-d(GGGTGGGTGGGTGGGT) (T30695) and 5-d(GTGGTGGGTGGGTGGGT) (T30177) from extremely stable intramolecular G-tetrads via a two-step process that involves the binding of one K+ ion to a central pair of G-quartets and two additional K+ ions, presumably, to the loops (Jing et al., (1997) Biochemistry in press). In that these oligonucleotides are potent HIV-1 inhibitors and among the most active HIV-1 integrase inhibitors yet identified, we have sought to further characterize the K(+)-induced folding process for the purpose of rational chemical modification of these anti-HIV agents. Our NMR investigation demonstrates that in the presence of Li+ ions, T30695 forms an unimolecular tetrad fold, stabilized by a pair of syn-anti-syn-anti G-quartets comprising a central core. The NMR spectrum of T30695 as a function of K+ titration reveals a well-defined transition that saturates upon addition of three K+ ions per oligomer. During this process, the initial Li(+)-dependent G-quartet structure converts into a highly symmetrical, stable form (the NMR detected melting transition temperature is increased by approximately 20 degrees C). The conformation of the G-quartet core remains unchanged, while the loosely structured loop residues become organized in a fashion which is stabilized by K+ ion binding and by interactions with the core. To explain these data, we propose a model wherein K+ binding to the loops induces structural rearrangement, to yield a planar array of loop bases in proximity to the underlying G-quartets. By reference to closely related homologues, which lack activity as an HIV-1 or integrase inhibitor, the possibility is discussed that this ion-coordinated loop structure is crucial to the biological activity of T30695.

An intensely luminescent polymeric lanthanide chelator for multiple fluorescence labeling of biomolecules

Abstract A new bifunctional chelating agent has been synthesized and used to prepare a stable, intensely luminescent polymer, PLCS-Eu(III), with an extinction coefficient of 10 +6 (M-cm) −1 , a very large stokes shift (Ex 337, Em 617nm), a 5nm emission bandwidth and a quantum yield of 0.6. The PLCS-Eu(III) complex was found stable towards EDTA and prolonged irradiation. It was coupled, as a coating, upon 3μm beads which in turn were coupled to oligonucleotides, for use in a nucleic acid sandwich assay. Its millisecond emission lifetime will allow it to be used for time resolved fluorescence detection. PLCS-Eu(III), a novel chelator, may be a useful new tool for array based studies of biopolymer interactions.

Synthesis of Triple Helix Forming Oligonucleotides Containing 2′-Deoxyformycin A

Abstract N7-Benzoyl-2′-deoxyformycin A (5) was prepared from formycin A and incorporated into the triple helix forming oligonucleotide PRE2ap at CG inversion sites. The modified oligonucleotide containing three substitutions of 2′-deoxyformycin A displayed a 10-fold increase in binding affinity as compared to its unmodified counterpart. This provided a method to accommodate CG inversion sites within target sites for antiparallel triple helix formation.

Genosensors: microfabricated devices for automated DNA sequence analysis

A new technology is introduced for developing potentially low cost, high throughput DNA sequence analysis. This approach utilizes novel bioelectronic genosensor devices to rapidly detect hybridization events across a DNA probe array. Detection of DNA probe/target hybridization has been achieved by two electronic methods. The first method utilizes a permittivity chip which interrogates the miniature test fixtures with a low voltage alternating electric field. The second method, which is the emphasis of this paper, utilizes a charge- coupled device (CCD) to detect the hybridization of appropriately tagged (radioisotope, fluorescent, or chemiluminescent labels) target DNA to an array of DNA probes immobilized above the pixels. Such direct electronic-biologic coupling is shown to provide a tenfold sensitivity improvement over conventional lens-based detection systems.

A Non-Watson–Crick Motif of Base-pairing on Surfaces for Untethered Oligonucleotides

A structural view of DNA association/hybridization to a target oligonucleotide molecule near a surface has been developed. Recent experiments have showed a kinetically rapid hybridization between large target DNA fragments and oligonucleotides electrostatically immobilized (untethered) to a surface. Theory and computer simulations have been used to investigate the nature of the specificity and affinity in such a system. Simulations were performed for a modified silicon dioxide surface with positively charged groups at neutral pH. The dosing of a surface with unattached oligonucleotide was simulated. The oligonucleotide was found to associate with the surface in salt water in a way that some of the bases remained stacked, and most of the bases near the surface on average pointed preferentially toward the solution, away from the surface. Use of an analytic solution to the linear Poisson–Boltzmann (PB) theory of the electric double layer interaction between DNA and a hard surface predicts tight binding in this system. The simulation thus gives a mechanism for specificity and the theory a mechanism for affinity. The geometry is such that only non-helical base pairs would be accommodated with an irregular backbone.

Advanced approach to simultaneous monitoring of multiple bacteria in space

The utility of a novel microarray-based microbial analyzer was demonstrated by the rapid detection, imaging, and identification of a mixture of microorganisms found in a waste water sample from the Lunar-Mars Life Support Test Project through the synergistic combination of: (1) judicious RNA probe selection via algorithms developed by University of Houston scientists; (2) tuned surface chemistries developed by Baylor College of Medicine scientists to facilitate hybridization of rRNA targets to DNA probes under very low salt conditions, thereby minimizing secondary structure; and (3) integration of the microarray printing and detection/imaging instrumentation by Genometrix to complete the quantitative analysis of microorganism mixtures.

Solution structure of an antiparallel purine motif triplex containing a T·CG pyrimidine base triple

BACKGROUNDnTriplex formation is an approach of potential use in regulating and mapping of gene sequences. However, such applications have been limited to homogeneous sequences consisting of stretches of purines or pyrimidines. Understanding how heterogeneous duplexes are recognized by a third strand oligonucleotide at the atomic resolution level is an essential step toward broadening the application of triplex formation into biochemical and biomedical areas.nnnRESULTSnThe solution structure of an antiparallel triplex (RRY6) containing a site of inversion (i.e. a T within a homopurine stretch, forming a T.CG base triple) has been determined using NMR-restrained computations in the presence of explicit water. The results reveal that within the RRY6 triplex the conformation of the duplex is mostly B-like and that of the third strand exhibits significant variations in interbase separations and backbone torsion angles. A major displacement of the inversion site T sugar in a 5-direction, accompanied by the tilt of the T base in T.CG, was observed. The T.CG base triple contains a single hydrogen bond between T O4 and the exposed C amino proton and is stabilized by a number of interstrand and sequential van der Waal contacts. The structural comparisons of RRY6 with two related triplexes indicate localized perturbation at the non-classical base triple site. Various triplexes contain sugars in the C2-endo family and the global features of their duplexes are similar.nnnCONCLUSIONSnThis study provides valuable information concerning the molecular basis of the specific recognition of a Watson-Crick base paired C residue at the inversion sites in the antiparallel triplex and should lead to general rules for designing triplexes containing heterogeneous sequences.

Triplex formation at the rat neu oncogene promoter

Current cancer chemotherapy treatments generally act by affecting rapidly growing malignant cells. Unfortunately, they are relatively nonspecific and thus have a tendency to affect other rapidly growing normal cells in a deleterious manner. Triplex-forming oligodeoxyribonucleotides (TFOs) promise to be a new class of sequence-specific DNA-binding drugs which will target malignancies at the transcriptional level. The formation of an intermolecular triplex (triple helix) has been shown to block the binding of transcription factors and repress transcription in genes such as c-myc and that encoding the epidermal growth factor receptor. The rat neu oncogene promoter contain promoter-enhancer elements which are purine/pyrimidine rich. These enhancer elements are amenable to targeting by TFOs. the human counterpart of rat neu, HER2, is often found to be amplified or overexpressed in a variety of malignancies, such as those of the breast, lungs, ovary, colon and stomach. TFOs may proved to be the basis of effective chemotherapy drugs for these cancers. TFO binding at the GTG element (5GGTGGGGGGG) and at the GA element (5GGAGGAGGAGGG) has been characterized by gel mobility shift analysis and DNase 1 footprinting. Binding has been shown to occur at a Kd as low as 10(-8) M and has been shown to be sequence specific.