Richard Fahlman - Academia.edu (original) (raw)
Papers by Richard Fahlman
Nano Letters, Jun 28, 2003
A versatile role for guanine−guanine mismatches within DNA double helices such as the formation o... more A versatile role for guanine−guanine mismatches within DNA double helices such as the formation of specific, interhelical synaptic events has previously been reported. Here, we demonstrate that certain categories of guanine−guanine mismatches within double helices enable a different structural/conformational transitionsa reversible, intramolecular "pinching" of the duplex, initiated and reversed by the binding and dissociation of certain specific cations. Such a "pinching" transition should provide a valuable "contractile" feature to the design of new, DNA-based molecular motors and nanoconstructions.
Nucleic Acids Research, Dec 18, 2008
Although some experiments suggest that the ribosome displays specificity for the identity of the ... more Although some experiments suggest that the ribosome displays specificity for the identity of the esterified amino acid of its aminoacyl-tRNA substrate, a study measuring dissociation rates of several misacylated tRNAs containing the GAC anticodon from the A site showed little indication for such specificity. In this article, an expanded set of misacylated tRNAs and two 2'-deoxynucleotidesubstituted mRNAs are used to demonstrate the presence of a lower threshold in k off values for aa-tRNA binding to the A site. When a tRNA binds sufficiently well to reach this threshold, additional stabilizing effects due to the esterified amino acid or changes in tRNA sequence are not observed. However, specificity for different amino acid side chains and the tRNA body is observed when tRNA binding is sufficiently weaker than this threshold. We propose that uniform aa-tRNA binding to the A site may be a consequence of a conformational change in the ribosome, induced by the presence of the appropriate combination of contributions from the anticodon, amino acid and tRNA body.
Journal of the American Chemical Society, Nov 17, 1999
Much recent interest has focused on DNA as a material for the construction of two-and threedimens... more Much recent interest has focused on DNA as a material for the construction of two-and threedimensional objects on the nanometer to micrometer scale. Such constructions have made use of the recognition of "complementary" nucleotide sequence by single-stranded stretches of DNA and the formation of double helices (duplexes) via the formation of Watson-Crick base pairs. Recently, we have described a quite distinct paradigm for the side-by-side binding ("synapsis") by two intact DNA duplexes modified by the inclusion of short elements of eight guanine-guanine mismatch base pairs (a "G-G domain") within them. Here, we demonstrate that it is possible to design nonidentical G-G domains, which have the property of synapsing exclusively to "self" as opposed to "non-self". Two modes of synapsis are observed in this versatile system: that in which two distinct species of duplex in a mixture concertedly undergo "self"-synapsis and that in which one designated species out of the two "self"-synapses specifically. Incorporation of these novel methodologies for "self"-synapsis and site-specific synapsis by DNA duplexes into current methods for constructing DNA nanostructures and mesostructures may facilitate the assembly of more complex DNAbased materials and arrays.
Journal of Molecular Biology, Jul 1, 1998
The simple innovation of introducing a block of G ÁG mismatches into a Watson-Crick DNA duplex pe... more The simple innovation of introducing a block of G ÁG mismatches into a Watson-Crick DNA duplex permits two such duplexes, under conditions of physiological temperature and salt, to``synapse'' with one another at their G ÁG mismatch sites via guanine-quartet formation. The short quadruplex formed at the``synapsed'' site necessarily has its strands in an antiparallel, or partially antiparallel orientation. We wished to test whether a different, and more stable, synapsis might be achieved if one of the two strands in the synapsable duplex had its domain of guanine residues in a reverse orientation to the rest of the strand, via 5 H-5 H and 3 H-3 H linkages. Such modi®ed duplexes might synapse via the formation of the thermodynamically preferred parallel quadruplex. Our results indicate that such``parallel'' and``antiparallel'' synaptic events have dramatically different requirements for cations. We use chemical probing experiments to provide evidence for a kinetic model for this discrepancy. It may be possible to exploit the distinct properties of the above two kinds of synapsable duplexes for a variety of in vivo and in vitro applications.
Journal of the American Chemical Society, Apr 2, 2002
The electrical conductivity of DNA is dependent on its conformational state. We demonstrate here ... more The electrical conductivity of DNA is dependent on its conformational state. We demonstrate here that such a dependence may be harnessed for the electronic sensing of external analytes, for instance, adenosine. Such a DNA sensor incorporates an analyte "receptor", whose altered conformation in the presence of bound analyte switches the conformation, and hence, the conductive path between two DNA double-helical stems. Two distinct designs for such sensors are described here, that permit significant electrical conduction through a "detector" double-helical stem only in the presence of the bound analyte. In the first design, current flows through the analyte receptor itself, whereas in the second, current flows in a path adjacent to the receptor. The former design may be especially suitable for certain categories of analytes, including heterocycle-containing compounds such as adenosine, whereas the latter design should be generally applicable to the detection of any molecular analyte, large or small. Since analyte detection in these DNA sensors is electronic, the potential exists for their application in rapid and automated chipbased detection of small molecules as well as of proteins and other macromolecules.
Journal of Magnetic Resonance
Redox Biology
Tumour hypoxia negatively impacts therapy outcomes and continues to be a major unsolved clinical ... more Tumour hypoxia negatively impacts therapy outcomes and continues to be a major unsolved clinical problem. Nitroimidazoles are hypoxia selective compounds that become entrapped in hypoxic cells by forming drug-protein adducts. They are widely used as hypoxia diagnostics and have also shown promise as hypoxia-directed therapeutics. However, little is known about the protein targets of nitroimidazoles and the resulting effects of their modification on cancer cells. Here, we report the synthesis and applications of azidoazomycin arabinofuranoside (N3-AZA), a novel click-chemistry compatible 2-nitroimidazole, designed to facilitate (a) the LC-MS/MS-based proteomic analysis of 2-nitroimidazole targeted proteins in FaDu head and neck cancer cells, and (b) rapid and efficient labelling of hypoxic cells and tissues. Bioinformatic analysis revealed that many of the 62 target proteins we identified participate in key canonical pathways including glycolysis and HIF1A signaling that play critical roles in the cellular response to hypoxia. Critical cellular proteins such as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the detoxification enzyme glutathione S-transferase P (GSTP1) appeared as top hits, and N3-AZA adduct formation significantly reduced their enzymatic activities only under hypoxia. Therefore, GAPDH, GSTP1 and other proteins reported here may represent candidate targets to further enhance the potential for nitroimidazole-based cancer therapeutics.
Comparative Proteomics: Current capabilities and comparative analysis of tumor response to chemot... more Comparative Proteomics: Current capabilities and comparative analysis of tumor response to chemotherapy.
RNA (New York, N.Y.), Jun 21, 2017
Proteins of the Sm and Sm-like (LSm) families, referred to collectively as (L)Sm proteins, are fo... more Proteins of the Sm and Sm-like (LSm) families, referred to collectively as (L)Sm proteins, are found in all three domains of life, and are known to promote a variety of RNA processes such as base-pair formation, unwinding, RNA degradation, and RNA stabilization. In eukaryotes, (L)Sm proteins have been studied, inter alia, for their role in pre-mRNA splicing. In many organisms, the LSm proteins form two distinct complexes, one consisting of LSm1-7 that is involved in mRNA degradation in the cytoplasm, and the other consisting of LSm2-8 that binds spliceosomal U6 snRNA in the nucleus. We recently characterized the splicing proteins from the red alga Cyanidioschyzon merolae and found that it has only seven LSm proteins. The identities of CmLSm2-CmLSm7 were unambiguous, but the seventh protein was similar to LSm1 and LSm8. Here, we use in vitro binding measurements, microscopy, and affinity purification-mass spectrometry to demonstrate a canonical splicing function for the C. merolae LS...
Nucleic Acids Research, 2008
Although some experiments suggest that the ribosome displays specificity for the identity of the ... more Although some experiments suggest that the ribosome displays specificity for the identity of the esterified amino acid of its aminoacyl-tRNA substrate, a study measuring dissociation rates of several misacylated tRNAs containing the GAC anticodon from the A site showed little indication for such specificity. In this article, an expanded set of misacylated tRNAs and two 2'-deoxynucleotidesubstituted mRNAs are used to demonstrate the presence of a lower threshold in k off values for aa-tRNA binding to the A site. When a tRNA binds sufficiently well to reach this threshold, additional stabilizing effects due to the esterified amino acid or changes in tRNA sequence are not observed. However, specificity for different amino acid side chains and the tRNA body is observed when tRNA binding is sufficiently weaker than this threshold. We propose that uniform aa-tRNA binding to the A site may be a consequence of a conformational change in the ribosome, induced by the presence of the appropriate combination of contributions from the anticodon, amino acid and tRNA body.
Nature Structural & Molecular Biology, 2005
The binding of seven tRNA anticodons to their complementary codons on Escherichia coli ribosomes ... more The binding of seven tRNA anticodons to their complementary codons on Escherichia coli ribosomes was substantially impaired, as compared with the binding of their natural tRNAs, when they were transplanted into tRNA 2 Ala. An analysis of chimeras composed of tRNA 2 Ala and various amounts of either tRNA 3 Gly or tRNA 2 Arg indicates that the presence of the parental 32-38 nucleotide pair is sufficient to restore ribosome binding of the transplanted anticodons. Furthermore, mutagenesis of tRNA 2 Ala showed that its highly conserved A32-U38 pair serves to weaken ribosome affinity. We propose that this negative binding determinant is used to offset the very tight codon-anticodon interaction of tRNA 2 Ala. This suggests that each tRNA sequence has coevolved with its anticodon to tune ribosome affinity to a value that is the same for all tRNAs.
Nano Letters, 2003
A versatile role for guanine−guanine mismatches within DNA double helices such as the formation o... more A versatile role for guanine−guanine mismatches within DNA double helices such as the formation of specific, interhelical synaptic events has previously been reported. Here, we demonstrate that certain categories of guanine−guanine mismatches within double helices enable a different structural/conformational transitionsa reversible, intramolecular "pinching" of the duplex, initiated and reversed by the binding and dissociation of certain specific cations. Such a "pinching" transition should provide a valuable "contractile" feature to the design of new, DNA-based molecular motors and nanoconstructions.
Journal of the American Chemical Society, 1999
Much recent interest has focused on DNA as a material for the construction of two-and threedimens... more Much recent interest has focused on DNA as a material for the construction of two-and threedimensional objects on the nanometer to micrometer scale. Such constructions have made use of the recognition of "complementary" nucleotide sequence by single-stranded stretches of DNA and the formation of double helices (duplexes) via the formation of Watson-Crick base pairs. Recently, we have described a quite distinct paradigm for the side-by-side binding ("synapsis") by two intact DNA duplexes modified by the inclusion of short elements of eight guanine-guanine mismatch base pairs (a "G-G domain") within them. Here, we demonstrate that it is possible to design nonidentical G-G domains, which have the property of synapsing exclusively to "self" as opposed to "non-self". Two modes of synapsis are observed in this versatile system: that in which two distinct species of duplex in a mixture concertedly undergo "self"-synapsis and that in which one designated species out of the two "self"-synapses specifically. Incorporation of these novel methodologies for "self"-synapsis and site-specific synapsis by DNA duplexes into current methods for constructing DNA nanostructures and mesostructures may facilitate the assembly of more complex DNAbased materials and arrays.
Journal of the American Chemical Society, 2002
The electrical conductivity of DNA is dependent on its conformational state. We demonstrate here ... more The electrical conductivity of DNA is dependent on its conformational state. We demonstrate here that such a dependence may be harnessed for the electronic sensing of external analytes, for instance, adenosine. Such a DNA sensor incorporates an analyte "receptor", whose altered conformation in the presence of bound analyte switches the conformation, and hence, the conductive path between two DNA double-helical stems. Two distinct designs for such sensors are described here, that permit significant electrical conduction through a "detector" double-helical stem only in the presence of the bound analyte. In the first design, current flows through the analyte receptor itself, whereas in the second, current flows in a path adjacent to the receptor. The former design may be especially suitable for certain categories of analytes, including heterocycle-containing compounds such as adenosine, whereas the latter design should be generally applicable to the detection of any molecular analyte, large or small. Since analyte detection in these DNA sensors is electronic, the potential exists for their application in rapid and automated chipbased detection of small molecules as well as of proteins and other macromolecules.
Journal of Molecular Biology, 1998
The simple innovation of introducing a block of G ÁG mismatches into a Watson-Crick DNA duplex pe... more The simple innovation of introducing a block of G ÁG mismatches into a Watson-Crick DNA duplex permits two such duplexes, under conditions of physiological temperature and salt, to``synapse'' with one another at their G ÁG mismatch sites via guanine-quartet formation. The short quadruplex formed at the``synapsed'' site necessarily has its strands in an antiparallel, or partially antiparallel orientation. We wished to test whether a different, and more stable, synapsis might be achieved if one of the two strands in the synapsable duplex had its domain of guanine residues in a reverse orientation to the rest of the strand, via 5 H-5 H and 3 H-3 H linkages. Such modi®ed duplexes might synapse via the formation of the thermodynamically preferred parallel quadruplex. Our results indicate that such``parallel'' and``antiparallel'' synaptic events have dramatically different requirements for cations. We use chemical probing experiments to provide evidence for a kinetic model for this discrepancy. It may be possible to exploit the distinct properties of the above two kinds of synapsable duplexes for a variety of in vivo and in vitro applications.
Journal of the American Chemical Society, 2002
The mechanism for electrical charge conduction in DNA has been the subject of much recent interes... more The mechanism for electrical charge conduction in DNA has been the subject of much recent interest and debate. Many of the measurements of DNA conductivity have been made in aqueous solution, with an aromatic photooxidant moiety such as anthraquinone or a rhodium(III) complex covalently tethered to the DNA. Such studies, however, have given discrepant results, for instance, regarding the relative ability of AT-and GC-rich sequences to conduct charge and the possibility of thymine cyclobutane dimer repair through the DNA from a distance. A recent paper on conduction in DNA immobile four-way junctions using the rhodium photooxidant reported conduction in all four helical arms, contrary to what is known about the three-dimensional structure and stacking of 4-way junctions. We have reexamined conduction in such junctions using rhodium [Rh(phi) 2(byp*)Cl3] as well as the anthraquinone photooxidants, and find that although our rhodium data agree with the previously published work, the anthraquinone data reveal conduction in only two of the four helical arms, consistent with the known tertiary structure of four-way junctions. An electrophoretic investigation revealed the formation of intermolecular aggregates in the rhodiumderivatized junctions, but not in the anthraquinone-labeled junctions. Rhodium-specific aggregation was also observed with simple DNA duplexes under the same experimental conditions. A characteristic property of aggregation was that all participating DNA molecules required the rhodium derivatization, and underivatized molecules did not aggregate with the derivatized ones. It is conceivable that the results reported here will help reconcile the various discrepancies that have been reported from charge conduction experiments carried out on DNA utilizing different photooxidants.
Canadian Urological Association journal = Journal de l'Association des urologues du Canada
Partial bladder outlet obstruction (pBOO) is a ubiquitous problem in urology. From posterior uret... more Partial bladder outlet obstruction (pBOO) is a ubiquitous problem in urology. From posterior urethral valves to prostatic hypertrophy, pBOO results in significant morbidity and mortality. However, the pathophysiology is not completely understood. Proteomics uses mass spectrometry to accurately quantify change in tissue protein concentration. Therefore, we have applied proteomic analysis to a rodent model to assess for protein changes after a surgically induced pBOO. We hypothesize that proteomic analysis after an acute obstruction will determine the most prevalent initial protein response and, potentially, novel molecular pathways. Sprague Dawley rats underwent a surgically induced pBOO (n = 3 per group) for 3, 7, or 14 days. Bladders were assessed for weight and urodynamic parameters. Proteomics used liquid-chromatography based mass spectrometry. Polymerase chain reaction (PCR) was performed on tissue samples to confirm increased mRNA transcription. Bladder weight and capacity incr...
Nano Letters, Jun 28, 2003
A versatile role for guanine−guanine mismatches within DNA double helices such as the formation o... more A versatile role for guanine−guanine mismatches within DNA double helices such as the formation of specific, interhelical synaptic events has previously been reported. Here, we demonstrate that certain categories of guanine−guanine mismatches within double helices enable a different structural/conformational transitionsa reversible, intramolecular "pinching" of the duplex, initiated and reversed by the binding and dissociation of certain specific cations. Such a "pinching" transition should provide a valuable "contractile" feature to the design of new, DNA-based molecular motors and nanoconstructions.
Nucleic Acids Research, Dec 18, 2008
Although some experiments suggest that the ribosome displays specificity for the identity of the ... more Although some experiments suggest that the ribosome displays specificity for the identity of the esterified amino acid of its aminoacyl-tRNA substrate, a study measuring dissociation rates of several misacylated tRNAs containing the GAC anticodon from the A site showed little indication for such specificity. In this article, an expanded set of misacylated tRNAs and two 2'-deoxynucleotidesubstituted mRNAs are used to demonstrate the presence of a lower threshold in k off values for aa-tRNA binding to the A site. When a tRNA binds sufficiently well to reach this threshold, additional stabilizing effects due to the esterified amino acid or changes in tRNA sequence are not observed. However, specificity for different amino acid side chains and the tRNA body is observed when tRNA binding is sufficiently weaker than this threshold. We propose that uniform aa-tRNA binding to the A site may be a consequence of a conformational change in the ribosome, induced by the presence of the appropriate combination of contributions from the anticodon, amino acid and tRNA body.
Journal of the American Chemical Society, Nov 17, 1999
Much recent interest has focused on DNA as a material for the construction of two-and threedimens... more Much recent interest has focused on DNA as a material for the construction of two-and threedimensional objects on the nanometer to micrometer scale. Such constructions have made use of the recognition of "complementary" nucleotide sequence by single-stranded stretches of DNA and the formation of double helices (duplexes) via the formation of Watson-Crick base pairs. Recently, we have described a quite distinct paradigm for the side-by-side binding ("synapsis") by two intact DNA duplexes modified by the inclusion of short elements of eight guanine-guanine mismatch base pairs (a "G-G domain") within them. Here, we demonstrate that it is possible to design nonidentical G-G domains, which have the property of synapsing exclusively to "self" as opposed to "non-self". Two modes of synapsis are observed in this versatile system: that in which two distinct species of duplex in a mixture concertedly undergo "self"-synapsis and that in which one designated species out of the two "self"-synapses specifically. Incorporation of these novel methodologies for "self"-synapsis and site-specific synapsis by DNA duplexes into current methods for constructing DNA nanostructures and mesostructures may facilitate the assembly of more complex DNAbased materials and arrays.
Journal of Molecular Biology, Jul 1, 1998
The simple innovation of introducing a block of G ÁG mismatches into a Watson-Crick DNA duplex pe... more The simple innovation of introducing a block of G ÁG mismatches into a Watson-Crick DNA duplex permits two such duplexes, under conditions of physiological temperature and salt, to``synapse'' with one another at their G ÁG mismatch sites via guanine-quartet formation. The short quadruplex formed at the``synapsed'' site necessarily has its strands in an antiparallel, or partially antiparallel orientation. We wished to test whether a different, and more stable, synapsis might be achieved if one of the two strands in the synapsable duplex had its domain of guanine residues in a reverse orientation to the rest of the strand, via 5 H-5 H and 3 H-3 H linkages. Such modi®ed duplexes might synapse via the formation of the thermodynamically preferred parallel quadruplex. Our results indicate that such``parallel'' and``antiparallel'' synaptic events have dramatically different requirements for cations. We use chemical probing experiments to provide evidence for a kinetic model for this discrepancy. It may be possible to exploit the distinct properties of the above two kinds of synapsable duplexes for a variety of in vivo and in vitro applications.
Journal of the American Chemical Society, Apr 2, 2002
The electrical conductivity of DNA is dependent on its conformational state. We demonstrate here ... more The electrical conductivity of DNA is dependent on its conformational state. We demonstrate here that such a dependence may be harnessed for the electronic sensing of external analytes, for instance, adenosine. Such a DNA sensor incorporates an analyte "receptor", whose altered conformation in the presence of bound analyte switches the conformation, and hence, the conductive path between two DNA double-helical stems. Two distinct designs for such sensors are described here, that permit significant electrical conduction through a "detector" double-helical stem only in the presence of the bound analyte. In the first design, current flows through the analyte receptor itself, whereas in the second, current flows in a path adjacent to the receptor. The former design may be especially suitable for certain categories of analytes, including heterocycle-containing compounds such as adenosine, whereas the latter design should be generally applicable to the detection of any molecular analyte, large or small. Since analyte detection in these DNA sensors is electronic, the potential exists for their application in rapid and automated chipbased detection of small molecules as well as of proteins and other macromolecules.
Journal of Magnetic Resonance
Redox Biology
Tumour hypoxia negatively impacts therapy outcomes and continues to be a major unsolved clinical ... more Tumour hypoxia negatively impacts therapy outcomes and continues to be a major unsolved clinical problem. Nitroimidazoles are hypoxia selective compounds that become entrapped in hypoxic cells by forming drug-protein adducts. They are widely used as hypoxia diagnostics and have also shown promise as hypoxia-directed therapeutics. However, little is known about the protein targets of nitroimidazoles and the resulting effects of their modification on cancer cells. Here, we report the synthesis and applications of azidoazomycin arabinofuranoside (N3-AZA), a novel click-chemistry compatible 2-nitroimidazole, designed to facilitate (a) the LC-MS/MS-based proteomic analysis of 2-nitroimidazole targeted proteins in FaDu head and neck cancer cells, and (b) rapid and efficient labelling of hypoxic cells and tissues. Bioinformatic analysis revealed that many of the 62 target proteins we identified participate in key canonical pathways including glycolysis and HIF1A signaling that play critical roles in the cellular response to hypoxia. Critical cellular proteins such as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the detoxification enzyme glutathione S-transferase P (GSTP1) appeared as top hits, and N3-AZA adduct formation significantly reduced their enzymatic activities only under hypoxia. Therefore, GAPDH, GSTP1 and other proteins reported here may represent candidate targets to further enhance the potential for nitroimidazole-based cancer therapeutics.
Comparative Proteomics: Current capabilities and comparative analysis of tumor response to chemot... more Comparative Proteomics: Current capabilities and comparative analysis of tumor response to chemotherapy.
RNA (New York, N.Y.), Jun 21, 2017
Proteins of the Sm and Sm-like (LSm) families, referred to collectively as (L)Sm proteins, are fo... more Proteins of the Sm and Sm-like (LSm) families, referred to collectively as (L)Sm proteins, are found in all three domains of life, and are known to promote a variety of RNA processes such as base-pair formation, unwinding, RNA degradation, and RNA stabilization. In eukaryotes, (L)Sm proteins have been studied, inter alia, for their role in pre-mRNA splicing. In many organisms, the LSm proteins form two distinct complexes, one consisting of LSm1-7 that is involved in mRNA degradation in the cytoplasm, and the other consisting of LSm2-8 that binds spliceosomal U6 snRNA in the nucleus. We recently characterized the splicing proteins from the red alga Cyanidioschyzon merolae and found that it has only seven LSm proteins. The identities of CmLSm2-CmLSm7 were unambiguous, but the seventh protein was similar to LSm1 and LSm8. Here, we use in vitro binding measurements, microscopy, and affinity purification-mass spectrometry to demonstrate a canonical splicing function for the C. merolae LS...
Nucleic Acids Research, 2008
Although some experiments suggest that the ribosome displays specificity for the identity of the ... more Although some experiments suggest that the ribosome displays specificity for the identity of the esterified amino acid of its aminoacyl-tRNA substrate, a study measuring dissociation rates of several misacylated tRNAs containing the GAC anticodon from the A site showed little indication for such specificity. In this article, an expanded set of misacylated tRNAs and two 2'-deoxynucleotidesubstituted mRNAs are used to demonstrate the presence of a lower threshold in k off values for aa-tRNA binding to the A site. When a tRNA binds sufficiently well to reach this threshold, additional stabilizing effects due to the esterified amino acid or changes in tRNA sequence are not observed. However, specificity for different amino acid side chains and the tRNA body is observed when tRNA binding is sufficiently weaker than this threshold. We propose that uniform aa-tRNA binding to the A site may be a consequence of a conformational change in the ribosome, induced by the presence of the appropriate combination of contributions from the anticodon, amino acid and tRNA body.
Nature Structural & Molecular Biology, 2005
The binding of seven tRNA anticodons to their complementary codons on Escherichia coli ribosomes ... more The binding of seven tRNA anticodons to their complementary codons on Escherichia coli ribosomes was substantially impaired, as compared with the binding of their natural tRNAs, when they were transplanted into tRNA 2 Ala. An analysis of chimeras composed of tRNA 2 Ala and various amounts of either tRNA 3 Gly or tRNA 2 Arg indicates that the presence of the parental 32-38 nucleotide pair is sufficient to restore ribosome binding of the transplanted anticodons. Furthermore, mutagenesis of tRNA 2 Ala showed that its highly conserved A32-U38 pair serves to weaken ribosome affinity. We propose that this negative binding determinant is used to offset the very tight codon-anticodon interaction of tRNA 2 Ala. This suggests that each tRNA sequence has coevolved with its anticodon to tune ribosome affinity to a value that is the same for all tRNAs.
Nano Letters, 2003
A versatile role for guanine−guanine mismatches within DNA double helices such as the formation o... more A versatile role for guanine−guanine mismatches within DNA double helices such as the formation of specific, interhelical synaptic events has previously been reported. Here, we demonstrate that certain categories of guanine−guanine mismatches within double helices enable a different structural/conformational transitionsa reversible, intramolecular "pinching" of the duplex, initiated and reversed by the binding and dissociation of certain specific cations. Such a "pinching" transition should provide a valuable "contractile" feature to the design of new, DNA-based molecular motors and nanoconstructions.
Journal of the American Chemical Society, 1999
Much recent interest has focused on DNA as a material for the construction of two-and threedimens... more Much recent interest has focused on DNA as a material for the construction of two-and threedimensional objects on the nanometer to micrometer scale. Such constructions have made use of the recognition of "complementary" nucleotide sequence by single-stranded stretches of DNA and the formation of double helices (duplexes) via the formation of Watson-Crick base pairs. Recently, we have described a quite distinct paradigm for the side-by-side binding ("synapsis") by two intact DNA duplexes modified by the inclusion of short elements of eight guanine-guanine mismatch base pairs (a "G-G domain") within them. Here, we demonstrate that it is possible to design nonidentical G-G domains, which have the property of synapsing exclusively to "self" as opposed to "non-self". Two modes of synapsis are observed in this versatile system: that in which two distinct species of duplex in a mixture concertedly undergo "self"-synapsis and that in which one designated species out of the two "self"-synapses specifically. Incorporation of these novel methodologies for "self"-synapsis and site-specific synapsis by DNA duplexes into current methods for constructing DNA nanostructures and mesostructures may facilitate the assembly of more complex DNAbased materials and arrays.
Journal of the American Chemical Society, 2002
The electrical conductivity of DNA is dependent on its conformational state. We demonstrate here ... more The electrical conductivity of DNA is dependent on its conformational state. We demonstrate here that such a dependence may be harnessed for the electronic sensing of external analytes, for instance, adenosine. Such a DNA sensor incorporates an analyte "receptor", whose altered conformation in the presence of bound analyte switches the conformation, and hence, the conductive path between two DNA double-helical stems. Two distinct designs for such sensors are described here, that permit significant electrical conduction through a "detector" double-helical stem only in the presence of the bound analyte. In the first design, current flows through the analyte receptor itself, whereas in the second, current flows in a path adjacent to the receptor. The former design may be especially suitable for certain categories of analytes, including heterocycle-containing compounds such as adenosine, whereas the latter design should be generally applicable to the detection of any molecular analyte, large or small. Since analyte detection in these DNA sensors is electronic, the potential exists for their application in rapid and automated chipbased detection of small molecules as well as of proteins and other macromolecules.
Journal of Molecular Biology, 1998
The simple innovation of introducing a block of G ÁG mismatches into a Watson-Crick DNA duplex pe... more The simple innovation of introducing a block of G ÁG mismatches into a Watson-Crick DNA duplex permits two such duplexes, under conditions of physiological temperature and salt, to``synapse'' with one another at their G ÁG mismatch sites via guanine-quartet formation. The short quadruplex formed at the``synapsed'' site necessarily has its strands in an antiparallel, or partially antiparallel orientation. We wished to test whether a different, and more stable, synapsis might be achieved if one of the two strands in the synapsable duplex had its domain of guanine residues in a reverse orientation to the rest of the strand, via 5 H-5 H and 3 H-3 H linkages. Such modi®ed duplexes might synapse via the formation of the thermodynamically preferred parallel quadruplex. Our results indicate that such``parallel'' and``antiparallel'' synaptic events have dramatically different requirements for cations. We use chemical probing experiments to provide evidence for a kinetic model for this discrepancy. It may be possible to exploit the distinct properties of the above two kinds of synapsable duplexes for a variety of in vivo and in vitro applications.
Journal of the American Chemical Society, 2002
The mechanism for electrical charge conduction in DNA has been the subject of much recent interes... more The mechanism for electrical charge conduction in DNA has been the subject of much recent interest and debate. Many of the measurements of DNA conductivity have been made in aqueous solution, with an aromatic photooxidant moiety such as anthraquinone or a rhodium(III) complex covalently tethered to the DNA. Such studies, however, have given discrepant results, for instance, regarding the relative ability of AT-and GC-rich sequences to conduct charge and the possibility of thymine cyclobutane dimer repair through the DNA from a distance. A recent paper on conduction in DNA immobile four-way junctions using the rhodium photooxidant reported conduction in all four helical arms, contrary to what is known about the three-dimensional structure and stacking of 4-way junctions. We have reexamined conduction in such junctions using rhodium [Rh(phi) 2(byp*)Cl3] as well as the anthraquinone photooxidants, and find that although our rhodium data agree with the previously published work, the anthraquinone data reveal conduction in only two of the four helical arms, consistent with the known tertiary structure of four-way junctions. An electrophoretic investigation revealed the formation of intermolecular aggregates in the rhodiumderivatized junctions, but not in the anthraquinone-labeled junctions. Rhodium-specific aggregation was also observed with simple DNA duplexes under the same experimental conditions. A characteristic property of aggregation was that all participating DNA molecules required the rhodium derivatization, and underivatized molecules did not aggregate with the derivatized ones. It is conceivable that the results reported here will help reconcile the various discrepancies that have been reported from charge conduction experiments carried out on DNA utilizing different photooxidants.
Canadian Urological Association journal = Journal de l'Association des urologues du Canada
Partial bladder outlet obstruction (pBOO) is a ubiquitous problem in urology. From posterior uret... more Partial bladder outlet obstruction (pBOO) is a ubiquitous problem in urology. From posterior urethral valves to prostatic hypertrophy, pBOO results in significant morbidity and mortality. However, the pathophysiology is not completely understood. Proteomics uses mass spectrometry to accurately quantify change in tissue protein concentration. Therefore, we have applied proteomic analysis to a rodent model to assess for protein changes after a surgically induced pBOO. We hypothesize that proteomic analysis after an acute obstruction will determine the most prevalent initial protein response and, potentially, novel molecular pathways. Sprague Dawley rats underwent a surgically induced pBOO (n = 3 per group) for 3, 7, or 14 days. Bladders were assessed for weight and urodynamic parameters. Proteomics used liquid-chromatography based mass spectrometry. Polymerase chain reaction (PCR) was performed on tissue samples to confirm increased mRNA transcription. Bladder weight and capacity incr...