Polymer Nanoarray Approach for the Characterization of Biomolecular Interactions.

Pair-wise interactions at the single-molecule level can be done with nanoprobing techniques, such as AFM force spectroscopy, optical tweezers, and magnetic tweezers. These techniques can be used to probe interactions between well-characterized assemblies of biomolecules, such as monomer-dimer, dimer-dimer, and trimer-monomer. An important step of these techniques is the proper assembly of dimers, trimers, and higher oligomers to enable the interactions to be probed. We have developed a novel approach in which a defined number of peptides are assembled along a flexible polymeric molecule that serves as a linear matrix, termed as flexible nanoarray (FNA). The construct is synthesized with the use of phosphoramidite chemistry (PA), in which non-nucleoside PA spacers and standard oligonucleotide synthesis are used to grow the polymeric chain with the desired length. The reactive sites are incorporated during FNA synthesis. As a result, the FNA polymer contains a set of predesigned reactive sites to which the peptides are covalently conjugated. We describe the protocol for the synthesis of FNA and the application of this methodology to measure the molecular interactions between amyloid peptides of monomer-monomer, monomer-dimer, and dimer-dimer.

Single-molecule probing of amyloid nano-ensembles using the polymer nanoarray approach.

Soluble amyloid-beta (Aß) oligomers are the prime causative agents of cognitive deficits during early stages of Alzheimer's disease (AD). The transient nature of the oligomers makes them difficult to characterize by traditional techniques, suggesting that advanced approaches are necessary. Previously developed fluorescence-based tethered approach for probing intermolecular interactions (TAPIN) and AFM-based single-molecule force spectroscopy are capable of probing dimers of Aß peptides. In this paper, a novel polymer nanoarray approach to probe trimers and tetramers formed by the Aß(14-23) segment of Aß protein at the single-molecule level is applied. By using this approach combined with TAPIN and AFM force spectroscopy, the impact of pH on the assembly of these oligomers was characterized. Experimental results reveal that pH affects the oligomer assembly process. At neutral pH, trimers and tetramers assemble into structures with a similar stability, while at acidic conditions (pH 3.7), the oligomers adopt a set of structures with different lifetimes and strengths. Models for the assembly of Aß(14-23) trimers and tetramers based on the results obtained is proposed.

Detection of Isoniazid-, Fluoroquinolone-, Amikacin-, and Kanamycin-Resistant Tuberculosis in an Automated, Multiplexed 10-Color Assay Suitable for Point-of-Care Use.

Extensively drug-resistant (XDR) tuberculosis (TB) cannot be easily or quickly diagnosed. We developed a rapid, automated assay for the detection of XDR-TB plus resistance to the drug isoniazid (INH) for point-of-care use. Using a simple filter-based cartridge with an integrated sample processing function, the assay identified a wide selection of wild-type and mutant sequences associated with XDR-TB directly from sputum. Four new large-Stokes-shift fluorophores were developed. When these four Stokes-shift fluorophores were combined with six conventional fluorophores, 10-color probe detection in a single PCR tube was enabled. A new three-phase, double-nested PCR approach allowed robust melting temperature analysis with enhanced limits of detection (LODs). Finally, newly designed sloppy molecular beacons identified many different mutations using a small number of probes. The assay correctly distinguished wild-type sequences from 32 commonly occurring mutant sequences tested in gyrA, gyrB, katG, and rrs genes and the promoters of inhA and eis genes responsible for resistance to INH, the fluoroquinolone (FQ) drugs, amikacin (AMK), and kanamycin (KAN). The LOD was 300 CFU of Mycobacterium tuberculosis in 1 ml sputum. The rate of detection of heteroresistance by the assay was equivalent to that by Sanger sequencing. In a blind study of 24 clinical sputum samples, resistance mutations were detected in all targets with 100% sensitivity, with the specificity being 93.7 to 100%. Compared to the results of phenotypic susceptibility testing, the sensitivity of the assay was 75% for FQs and 100% each for INH, AMK, and KAN and the specificity was 100% for INH and FQ and 94% for AMK and KAN. Our approach could enable testing for XDR-TB in point-of-care settings, potentially identifying highly drug-resistant TB more quickly and simply than currently available methods.

A Metal-free Click Chemistry Approach for the Assembly and Probing of Biomolecules.

Probing of biomolecular complexes by single-molecule force spectroscopy (SMFS) methods including AFM requires proper and suitable coupling methods for immobilization of biomolecules onto the AFM tip and the surface. The use of flexible tethers for the coupling process has dual advantages. First, they allow the specific immobilization of interacting molecules, and second, their flexibility facilitates the proper orientation of the interacting partners. Recently, we developed an approach termed Flexible Nano Array (FNA) in which interacting partners are located on the same polymeric FNA molecule separated by a flexible segment with a defined length. In this paper, we modified the FNA tether approach by incorporating click chemistry with non-metal modification. FNA was synthesized using DNA synthesis chemistry, in which phosphoramidite (PA) spacers containing six ethylene glycol units were used instead of nucleoside triphosphates. During the synthesis, two T modifiers conjugated to two dibenzocyclooctyl (DBCO) residues were incorporated at selected positions within the FNA. The DBCO functionality allows for coupling azide labeled biomolecules via click chemistry. Amyloid peptide Aß(14-23) terminated with azide was incorporated into the FNA and the reaction was controlled with mass-spectrometry. Assembly of tethered Aß(14-23) peptides into dimers was characterized by AFM force spectroscopy experiments in which the AFM tip functionalized with FNA terminated with biotin probed a streptavidin-coated mica surface. The formation of the peptide dimer was verified with force spectroscopy that showed the appearance of a specific fingerprint for dimer dissociation followed by a rupture event for the biotin-streptavidin link. The developed approach is capable of multiple probing events to allow the collection of a large set of data for a quantitative analysis of the force spectroscopy events.

A flexible nanoarray approach for the assembly and probing of molecular complexes.

Immobilization is a key step involved in probing molecular interactions using single-molecule force spectroscopy methods, including atomic force microscopy (AFM). To our knowledge, we describe a novel approach termed flexible nanoarray (FNA) in which the interaction between the two internally immobilized amyloid ß peptides is measured by pulling of the tether. The FNA tether was synthesized with nonnucleotide phosphoramidite monomers using the DNA synthesis chemistry. The two anchoring points for immobilization of the peptides inside the tether were incorporated at defined distances between them and from the ends of the polymer. Decamers of amyloid ß peptide capable of dimer formation were selected as a test system. The formation of the peptide dimers was verified by AFM force spectroscopy by pulling the tether at the ends. In these experiments, the thiolated end of the FNA tether was covalently immobilized on the AFM substrate functionalized with maleimide. The other end of the FNA tether was functionalized with biotin to form a noncovalent link with the streptavidin functionalized AFM tip during the approach stage. The dimers' rupture fingerprint was unambiguously identified on the force curves by its position and the force value. The FNA design allowed reversible experiments in which the monomers were allowed to associate after the rupture of the dimers by performing the approach stage before the rupture of the biotin-streptavidin link. This suggests that the FNA technique is capable of analyzing multiple intermolecular interactions in the same molecular complex. The computational analysis showed that the tethered peptides assemble into the same dimer structure as that formed by nontethered peptides, suggesting that the FNA tether has the necessary flexibility to enable assembly of the dimer even during the course of the force spectroscopy experiment.

Synthesis and physicochemical properties of 2'-deoxy-2',2''-difluoro-beta-D-ribofuranosyl and 2'-deoxy-2',2''-difluoro-alpha-D-ribofuranosyl oligonucleotides.

We present procedures for nucleoside and oligonucleotide synthesis, binding affinity (Tm) and structural analysis (CD spectra) of 2'-deoxy-2',2''-difluoro-alpha-D-ribofuranosyl and 2'-deoxy-2',2''-difluoro-beta-D-ribofuranosyl oligothymidylates. Possible reasons for the thermal instability of duplexes formed between these compounds and RNA or DNA targets are discussed.

A new synthesis of 9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)guanine (AraF-G).

Interesting and very promising antisense properties of 2'-deoxy-2'-fluoroarabinonucleic acids ((a) Wilds, C.J.; Damha, M.J. 2'-Deoxy-2'-fluoroarabinonucleosides and oligonucleotides (2'F-ANA): synthesis and physicochemical studies. Nucl. Acids Res. 2000, 28, 3625-3635; (b) Viazovkina, E.; Mangos, M.; Elzagheid, M.I.; Damha, M.J. Current Protocols in Nucleic Acid Chemistry 2002, 4.15.1-4.15.21) (2'F-ANA) has encouraged our research group to optimize the synthetic procedures for 2'-deoxy-2'-fluoro-beta-D-arabinonucleosides (araF-N). The synthesis of araF-U, araF-T, araF-A and araF-C is straightforward, (Tann, C.H.; Brodfuehrer, P.R.; Brundidge, S.P.; Sapino, C., Jr. Howell H.G. Fluorocarbohydrates in synthesis. An efficient synthesis of 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (beta-FIAU) and 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)thymine (beta-FMAU). J. Org. Chem. 1985, 50, 3644-3647; Howell, H.G.; Brodfuehrer, P.R.; Brundidge, S.P.; Benigni, D.A.; Sapino, C., Jr. Antiviral nucleosides. A stereospecific, total synthesis of 2'-fluoro-2'-deoxy-beta-D-arabinofuranosyl nucleosides. J. Org. Chem. 1988, 53, 85-88; Maruyama, T.; Takamatsu, S.; Kozai, S.; Satoh, Y.; Izana, K. Synthesis of 9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine bearing a selectively removable protecting group. Chem. Pharm. Bull. 1999, 47, 966-970) however, the synthesis of the guanine analogue is more complicated and affords poor to moderate yields of araF-G (4) ((a) Elzagheid, M.I.; Viazovkina, E.; Masad, M.J. Synthesis of protected 2'-deoxy-2'-fluoro-beta-D-arabinonucleosides. Synthesis of 2'-fluoroarabino nucleoside phosphoramidites and their use in the synthesis of 2'F-ANA. Current Protocols in Nucleic Acid Chemistry 2002, 1.7.1-1.7.19; (b) Tennila, T.; Azhayeva, E.; Vepsalainen, J.; Laatikainen, R.; Azhayev, A.; Mikhailopulo, I. Oligonucleotides containing 9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-adenine and -guanine: synthesis, hybridization and antisense properties. Nucleosides, Nucleotides and Nucl. Acids 2000, 19, 1861-1884). Here we describe an efficient synthesis of araF-G (4) that involves coupling of 2-deoxy-2-fluoro-3,5-di-O-benzoyl-alpha-D-arabinofuranosyl bromide (1) with 2-chlorohypoxanthine (2) to afford 2-chloro-beta-araF-I (3) in 52% yield. Nucleoside (3) was transformed into araF-G (4) by treatment with methanolic ammonia (150 degrees C, 6 h) in 67% yield.

Synthesis and properties of oligonucleotide chimeras containing 5'-amino-2'-deoxy-2'-fluoroarabinonucleosides.

Oligonucleotide analogues comprised of 2'-deoxy-2'-fluoro-beta-D-arabinose units joined via P3'-N5' phosphoramidate linkages (2'F-ANA(5'N)) were prepared for the first time. Among the compounds prepared were a series of 2'OMe-RNA-[GAP]-2'OMe-RNA 'chimeras', whereby the "GAP" consisted of DNA, DNA(5'N), 2'F-ANA or 2'F-ANA(5'N) segments. The chimeras with the 2'F-ANA and DNA gaps exhibited the highest affinity towards a complementary RNA target, followed by the 5'-amino derivatives, i.e., 2'F-ANA > DNA > 2'F-ANA(5'N) > DNA(5'N). Importantly, hybrids between these chimeras and target RNA were all substrates of both human RNase HII and E. coli RNase HI. In terms of efficiency of the chimera in recruiting the bacterial enzyme, the following order was observed: gap DNA > 2'F-ANA > 2'F-ANA(5'N) > DNA(5'N). The corresponding relative rates observed with the human enzyme were: gap DNA > 2'F-ANA(5'N) > 2'F-ANA > DNA(5'N).

Efficient RNase H-directed cleavage of RNA promoted by antisense DNA or 2'F-ANA constructs containing acyclic nucleotide inserts.

The ability of modified antisense oligonucleotides (AONs) containing acyclic interresidue units to support RNase H-promoted cleavage of complementary RNA is described. Manipulation of the backbone and sugar geometries in these conformationally labile monomers shows great benefits in the enzymatic recognition of the nucleic acid hybrids, while highlighting the importance of local strand conformation on the hydrolytic efficiency of the enzyme more conclusively. Our results demonstrate that the duplexes support remarkably high levels of enzymatic degradation when treated with human RNase HII, making them efficient mimics of the native substrates. Furthermore, interesting linker-dependent modulation of enzymatic activity is observed during in vitro assays, suggesting a potential role for this AON class in an RNase H-dependent pathway of controlling RNA expression. Additionally, the butyl-modified 2'F-ANA AONs described in this work constitute the first examples of a nucleic acid species capable of eliciting high RNase H activity while possessing a highly flexible molecular architecture at predetermined sites along the AON.

Antisense inhibition of Flk-1 by oligonucleotides composed of 2'-deoxy-2'-fluoro-beta-D-arabino- and 2'-deoxy-nucleosides.

The design of new antisense oligomers with improved binding affinity for targeted RNA, while still activating RNase H, is a major research area in medicinal chemistry. RNase H recognizes the RNA-DNA duplex and cleaves the complementary mRNA strand, providing the main mechanism by which antisense oligomers elicit their activities. It has been shown that configuration inversion at the C2' position of the DNA sugar moiety (arabinonucleic acid, ANA), combined with the substitution of the 2'OH group by a fluorine atom (2'F-ANA) increases the oligomer's binding affinity for targeted RNA. In the present study, we evaluated the antisense activity of mixed-backbone phosphorothioate oligomers composed of 2'-deoxy-2'-fluoro-beta-D-arabinose and 2'-deoxyribose sugars (S-2'F-ANA-DNA chimeras). We determined their abilities to inhibit the protein expression and phosphorylation of Flk-1, a vascular endothelial growth factor receptor (VEGF), and VEGF biological effects on endothelial cell proliferation, migration, and platelet-activating factor synthesis. Treatment of endothelial cells with chimeric oligonucleotides reduced Flk-1 protein expression and phosphorylation more efficiently than with phosphorothioate antisenses (S-DNA). Nonetheless, these two classes of antisenses inhibited VEGF activities equally. Herein, we also demonstrated the capacity of the chimeric oligomers to elicit RNase H activity and their improved binding affinity for complementary RNA as compared with S-DNA.

Oligonucleotides comprised of alternating 2'-deoxy-2'-fluoro-beta-D-arabinonucleosides and D-2'-deoxyribonucleosides (2'F-ANA/DNA 'altimers') induce efficient RNA cleavage mediated by RNase H.

Chimeric oligonucleotides comprised of alternating residues of 2'-deoxy-2'-fluoro-D-arabinonucleic acid (2'F-ANA) and DNA were synthesized and evaluated for an important antisense property-the ability to elicit ribonuclease H (RNase H) degradation of complementary RNA. Experiments used both human RNase HII and Escherichia coli RNase HI. Mixed backbone oligomers comprising alternating three-nucleotide segments of 2'F-ANA and three-nucleotide segments of DNA were the most efficient at eliciting RNase H degradation of target RNA, and were significantly better than oligonucleotides entirely composed of DNA, suggesting that these mixed backbone oligonucleotides may be potent antisense agents.

Potent gene-specific inhibitory properties of mixed-backbone antisense oligonucleotides comprised of 2'-deoxy-2'-fluoro-D-arabinose and 2'-deoxyribose nucleotides.

Phosphorothioate deoxyribonucleotides (PS-DNA) are among the most widely used antisense inhibitors. PS-DNA exhibits desirable properties such as enhanced nuclease resistance, improved bioavailability, and the ability to induce RNase H mediated degradation of target RNA. Unfortunately, PS-DNA possesses a relatively low binding affinity for target RNA that impacts on its potency in antisense applications. We recently showed that phosphodiester-linked oligonucleotides comprised of 2'-deoxy-2'-fluoro-D-arabinonucleic acid (FANA) exhibit both high binding affinity for target RNA and the ability to elicit RNase H degradation of target RNA [Damha et al. (1998) J. Am. Chem. Soc. 120, 12976]. In the present study, we evaluated the antisense activity of phosphorothioate-linked FANA oligonucleotides (PS-FANA). Oligonucleotides comprised entirely of PS-FANA were somewhat less efficient in directing RNase H cleavage of target RNA as compared to their phosphorothioate-linked DNA counterparts, and showed only weak antisense inhibition of cellular target expression. However, mixed-backbone oligomers comprised of PS-FANA flanking a central core of PS-DNA were found to possess potent antisense activity, inhibiting specific cellular gene expression with EC(50) values of less than 5 nM. This inhibition was a true antisense effect, as indicated by the dose-dependent decrease in both target protein and target mRNA. Furthermore, the appearance of mRNA fragments was consistent with RNase H mediated cleavage of the mRNA target. We also compared a series of PS-[FANA-DNA-FANA] mixed-backbone oligomers of varying PS-DNA core sizes with the corresponding 2'-O-methyl oligonucleotide chimeras, i.e., PS-[2'meRNA-DNA-2'meRNA]. Both types of oligomers showed very similar binding affinities toward target RNA. However, the antisense potency of the 2'-O-methyl chimeric compounds was dramatically attenuated with decreasing DNA core size, whereas that of the 2'-fluoroarabino compounds was essentially unaffected. Indeed, a PS-FANA oligomer containing a single deoxyribonucleotide residue core retained significant antisense activity. These findings correlated exactly with the ability of the various chimeric antisense molecules to elicit RNase H degradation of the target RNA in vitro, and suggest that this mode of inhibition is likely the most important determinant for potent antisense activity.

Synthesis of protected 2'-deoxy-2'-fluoro-beta-D-arabinonucleosides.

This unit describes in detail the preparation of protected 2'-deoxy-2'-fluoroarabinonucleosides. These building blocks are required for the synthesis of 2'-deoxy-2'-fluoroarabinonucleic acid (2'F-ANA), an oligonucleotide analog exhibiting very promising antisense properties. The preparation of phosphoramidites from these building blocks and the synthesis of 2'F-ANA are described elsewhere in the manual.

Solid-phase synthesis of 2'-deoxy-2'-fluoro- beta-D-oligoarabinonucleotides (2'F-ANA) and their phosphorothioate derivatives.

This unit describes the chemical synthesis of 2'-deoxy-2'-fluoro-b-D-oligoarabinonucleotides (2'F-ANA), both with phosphodiester and phosphorothioate linkages. The protocols described herein include araF phosphoramidite preparation, assembly on DNA synthesizers, and final deprotection and purification of oligonucleotides.