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Biomedical subjects

R L Letsinger

Publications and source records attributed to R L Letsinger.

At least 19 recordsLinked to original sources
PubMed2001

Dynamics of photoinduced charge transfer and hole transport in synthetic DNA hairpins.

The dynamics of photoinduced charge separation and charge recombination processes in synthetic DNA hairpins have been investigated by means of femtosecond transient absorption spectroscopy. The driving force and distance dependence of charge-transfer processes involving singlet acceptors and nucleobase donors are consistent with a single-step superexchange mechanism in which the electronic coupling between the donor and acceptor is strongly distance dependent. The dynamics of reversible hole transport between a primary guanine donor and nearby GG or GGG sequences has also been determined and establishes that these sequences are very shallow hole traps.

DNA↗
PubMed2000

A fluorescence-based method for determining the surface coverage and hybridization efficiency of thiol-capped oligonucleotides bound to gold thin films and nanoparticles.

Using a fluorescence-based method, we have determined the number of thiol-derivatized single-stranded oligonucleotides bound to gold nanoparticles and their extent of hybridization with complementary oligonucleotides in solution. Oligonucleotide surface coverages of hexanethiol 12-mer oligonucleotides on gold nanoparticles (34 +/- 1 pmol/cm2) were significantly higher than on planar gold thin films (18 +/- 3 pmol/cm2), while the percentage of hybridizable strands on the gold nanoparticles (1.3 +/- 0.3 pmol/cm2, 4%) was lower than for gold thin films (6 +/- 2 pmol/cm2, 33%). A gradual increase in electrolyte concentration over the course of oligonucleotide deposition significantly increases surface coverage and consequently particle stability. In addition, oligonucleotide spacer sequences improve the hybridization efficiency of oligonucleotide-modified nanoparticles from approximately 4 to 44%. The surface coverage of recognition strands can be tailored using coadsorbed diluent oligonucleotides. This provides a means of indirectly controlling the average number of hybridized strands per nanoparticle. The work presented here has important implications with regard to understanding interactions between modified oligonucleotides and metal nanoparticles, as well as optimizing the sensitivity of gold nanoparticle-based oligonucleotide detection methods.

Gold↗
PubMed2000

Scanometric DNA array detection with nanoparticle probes.

A method for analyzing combinatorial DNA arrays using oligonucleotide-modified gold nanoparticle probes and a conventional flatbed scanner is described here. Labeling oligonucleotide targets with nanoparticle rather than fluorophore probes substantially alters the melting profiles of the targets from an array substrate. This difference permits the discrimination of an oligonucleotide sequence from targets with single nucleotide mismatches with a selectivity that is over three times that observed for fluorophore-labeled targets. In addition, when coupled with a signal amplification method based on nanoparticle-promoted reduction of silver(I), the sensitivity of this scanometric array detection system exceeds that of the analogous fluorophore system by two orders of magnitude.

Base Pair Mismatch↗
PubMed1997

Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles.

A highly selective, colorimetric polynucleotide detection method based on mercaptoalkyloligonucleotide-modified gold nanoparticle probes is reported. Introduction of a single-stranded target oligonucleotide (30 bases) into a solution containing the appropriate probes resulted in the formation of a polymeric network of nanoparticles with a concomitant red-to-pinkish/purple color change. Hybridization was facilitated by freezing and thawing of the solutions, and the denaturation of these hybrid materials showed transition temperatures over a narrow range that allowed differentiation of a variety of imperfect targets. Transfer of the hybridization mixture to a reverse-phase silica plate resulted in a blue color upon drying that could be detected visually. The unoptimized system can detect about 10 femtomoles of an oligonucleotide.

Biosensing Techniques↗
PubMed1997

Distance-dependent electron transfer in DNA hairpins.

The distance dependence of photoinduced electron transfer in duplex DNA was determined for a family of synthetic DNA hairpins in which a stilbene dicarboxamide forms a bridge connecting two oligonucleotide arms. Investigation of the fluorescence and transient absorption spectra of these hairpins established that no photoinduced electron transfer occurs for a hairpin that has six deoxyadenosine-deoxythymidine base pairs. However, the introduction of a single deoxyguanosine-deoxycytidine base pair resulted in distance-dependent fluorescence quenching and the formation of the stilbene anion radical. Kinetic analysis suggests that duplex DNA is somewhat more effective than proteins as a medium for electron transfer but that it does not function as a molecular wire.

Base Composition↗
PubMed1996

A DNA-based method for rationally assembling nanoparticles into macroscopic materials.

Colloidal particles of metals and semiconductors have potentially useful optical, optoelectronic and material properties that derive from their small (nanoscopic) size. These properties might lead to applications including chemical sensors, spectroscopic enhancers, quantum dot and nanostructure fabrication, and microimaging methods. A great deal of control can now be exercised over the chemical composition, size and polydispersity of colloidal particles, and many methods have been developed for assembling them into useful aggregates and materials. Here we describe a method for assembling colloidal gold nanoparticles rationally and reversibly into macroscopic aggregates. The method involves attaching to the surfaces of two batches of 13-nm gold particles non-complementary DNA oligonucleotides capped with thiol groups, which bind to gold. When we add to the solution an oligonucleotide duplex with 'sticky ends' that are complementary to the two grafted sequences, the nanoparticles self-assemble into aggregates. This assembly process can be reversed by thermal denaturation. This strategy should now make it possible to tailor the optical, electronic and structural properties of the colloidal aggregates by using the specificity of DNA interactions to direct the interactions between particles of different size and composition.

Base Sequence↗
PubMed1996

Stabilization of triple-stranded oligonucleotide complexes: use of probes containing alternating phosphodiester and stereo-uniform cationic phosphoramidate linkages.

Pyrimidine oligonucleotides containing alternating anionic and stereo-uniform cationic N-(dimethylamino-propyl)phosphoramidate linkages [e.g. d(T+T-)7T, d(T+T-)2(T+C-)5T and (U'+U'-)7dT, where U' is 2'-O-methyluridine)] are shown to bind to complementary double-stranded DNA segments in 0.1 M NaCl at pH 7 to form triple-stranded complexes with the pyrimidine.purine.pyrimidine motif. For each of the sequences investigated, one stereoisomer bound with higher affinity, and the other stereoisomer with lower affinity, than the corresponding all-phosphodiester oligonucleotide. The stereoisomer of d(T+T-)7T that interacted weakly with a dT.dA target in 0.1 M NaCl formed a novel dA.dA.dT triple-stranded complex with poly(dA) or d(Al5C4A15) in 1 M NaCl; in contrast, the stereoisomer that bound strongly to the dT.dA target failed to form a dA.dA.dT triple-stranded complex.

DNA Probes↗
PubMed1996

Incorporation of a non-nucleotide bridge into hairpin oligonucleotides capable of high-affinity binding to the Rev protein of HIV-1.

A bridge containing a rigid trans-stilbene group, -P(O)(O-)O(CH2)3NHC(O)- C6H4-CH=CHC6H4C(O)NH(CH2)3OP(O)(O-)-, has been incorporated into several oligonucleotide sequences based on the minimal Rev Binding Element (RBE) of HIV-1. This bridge was found to be effective as a UUCG tetraloop in stabilizing short RNA duplex structures containing mismatched bases and bulged out nucleotide residues and to be more effective than either a TTTT loop or a triethyleneglycol linker in stabilizing similar DNA structures. Evaluation of stilbene-containing RNA RBE sequences of varying length for their ability to bind the Rev protein of HIV-1 showed that a 22-nucleotide stilbenedicarboxamide conjugate bound Rev almost as well as a 94-base fragment of the Rev Responsive Element (RRE). A DNA hairpin mimetic with the same sequence was incapable of Rev binding. Taken together, these experiments serve as an example for how in vitro selection and chemical modification can be combined to generate high-affinity mimetics of nucleic acid sequence and structure.

Base Sequence↗
PubMed1996

Stereochemical assignment of chiral phosphotriester analogues with Alu I sites.

The stereochemistry of the diastereomers of a DNA duplex with the 2,2,2-trichloro-1,1,-dimethylethyl (TCDME) phosphotriester backbone substitution has been assigned by the use of 2D NMR spectroscopy. The duplex [G1G2A3A4G5p(TCDME)C6T7A8G9G10]-[C11C12T13A14G15C16 T17T18C19C20] is a substrate of the restriction endonuclease Alu I, with placement of the TCDME group at the G5-C6 cleavage site of one strand. The stereochemical orientation of the TCDME group in relation to the structure of the double helix regulates the ability of Alu I to hydrolyze the complementary recognition site. The phosphotriester group of the isomer 1 duplex blocks cleavage of the complementary strand, while that of the isomer 2 duplex allows cleavage to proceed. Within the phosphotriester recognition site, no hydrolysis is detected nor is any seen when the single-stranded DNA substrate is utilized. Data from the 2D NOESY spectra demonstrate that both DNA duplexes retain basic B-form geometry. The isomer 1 duplex shows NOE cross-relaxation from the protons of the two methyl groups of the TCDME modification (1.99, 2.00 ppm) to the G5 H3'(5.30 ppm), G5 H4' (4.53 ppm), and C6 H5'/H5" (4.52, 4.62 ppm) protons. The isomer 2 duplex shows NOE cross-relaxation from the methyl protons of the TCDME modification (2.01, 2.03 ppm) to the C6 H6 (7.15 ppm), C6 H4' (4.30 ppm), C6 H5'/H5" (4.48, 4.62 ppm), G5 H3' (5.26 ppm), and G5 H4' (4.48 ppm) protons. Thus the NOE cross-relaxation between the methyl protons of the TCDME modification and the C6 H6 and C6 H4' protons in isomer 2 is not found in the spectra of the isomer 1 duplex. These NMR data confirm the stereochemical assignment of the chirality of the TCDME phosphotriester group in isomer 1 as the Sp configuration and in isomer 2 as the Rp configuration. The Sp isomer features the TCDME group pointing away from the helix, while the Rp isomer shows the TCDME group pointing towards the major groove. Thus through the use of 2D NMR techniques, the stereochemistry of chiral phosphotriester linkages may be assigned in chemically modified DNA.

Binding Sites↗
PubMed1994

Selective chemical autoligation on a double-stranded DNA template.

We show that a double-stranded DNA segment serves as an effective template for spontaneously coupling short pyrimidine oligonucleotides containing terminal -P(O)(O-)S- and BrCH2C(O)NH- groups. The efficiency of this autoligation depends markedly on proper base-pairing between the probe oligomers and the double-stranded target. This chemistry should be useful in designing highly selective probes for double-stranded polynucleotide segments.

Base Sequence↗
PubMed1994

Enhancement of selectivity in recognition of nucleic acids via chemical autoligation.

A new approach to increase the selectivity of interaction between oligonucleotide probes and target nucleic acids is described. In place of a single, relatively long oligonucleotide probe, two or three short oligomers terminated by thiophosphoryl and bromoacetamido groups are employed. Fast and efficient autoligation takes place when the oligomers hybridize in a contiguous mode to the same complementary strand such that a thiophosphoryl group on one strand and a bromoacetamido group on another are brought into proximity. A single nucleotide mismatch for the short probes leads to marked reduction in the rate of autoligation. The binding affinity of the product is close to that for a natural probe of the same length. This approach could have potential in oligonucleotide-based diagnostics, chemical amplification systems, and therapeutic applications.

Base Composition↗
PubMed1993

Template controlled coupling and recombination of oligonucleotide blocks containing thiophosphoryl groups.

Oxidation of a pair of 3'- and 5'-thiophosphoryloligonucleotides in the presence of a complementary oligonucleotide template is shown to provide an effective means for selectively linking oligonucleotide blocks. Coupling proceeds rapidly and efficiently under mild conditions in dilute aqueous solutions (microM range for oligomers, 2-15 min at 0-4 degrees C with K3Fe(CN)6 or KI3 as oxidant). This chemistry was demonstrated by polymerization of a thymidylate decamer derivative (sTTTTTTTTTTs) in the presence of poly(dA) and by coupling oligomers possessing terminal thiophosphoryl groups (ACACCCAATTs + sCTGAAAATGG and ACACCCAATs + sCTGAAAATGG) in the presence of a template (CCATTTTCAGAATTGGGTGT). Efficient linking of 5' to 3' phosphoryl groups can be achieved under conditions where virtually no coupling takes place in absence of a template. A novel feature of the chemistry is that catalyzed recombinations of oligomers containing internal -OP(O)(O-)SSP(O)(O-)O- linkages can be directed by hydrogen bonding to a complementary oligonucleotide. Convenient procedures are reported for solid phase synthesis of the requisite oligonucleotide 3'- and 5'-phosphorothioates.

Base Sequence↗
PubMed1992

Synthesis and properties of oligonucleotides containing aminodeoxythymidine units.

Procedures are described for synthesis via solid support methodology of oligonucleotide analogues derived in part from 3'-amino-3'-deoxythymidine or 5'-amino-5'-deoxythymidine. Oligothymidylate decamers terminated with a 3'-amino group or containing a 3'-NHP(O)(O-)O-5' internucleoside link are found to form unusually stable complexes with poly(dA), poly(A), and oligo(dA). For related derivatives of 5'-amino-5'-deoxythymidine enhancement is less or absent, and in the case of multiple substitution destabilization of the heteroduplex may be observed. That the effect of the 3'-amino group is general for oligonucleotide derivatives is indicated by enhanced Tm values for heteroduplex complexes of the mixed-base oligomer, d(TATTCAGTCAT(NH2)), and the methyl phosphonate derivatives, TmTmTmTmTmTmTmTmTmT(NH2) and d(TmAmTmTmCmAmGmTmCmAmT(NH2)).

Base Sequence↗
PubMed1992

Selective O-phosphitilation with nucleoside phosphoramidite reagents.

In contrast to tetrazole, pyridine hydrochloride/imidazole converts nucleoside phosphoramidites to intermediates that show a high preference for phosphitilating hydroxyl groups relative to nucleoside amino groups. Use of this activating agent and incorporation of a pyridine hydrochloride/aniline wash step in the synthetic cycles permit synthesis of mixed base twenty-mer oligonucleotides from nucleoside reagents containing unprotected amino groups. This approach should be useful for the synthesis of oligonucleotide analogues containing substituents sensitive to reagents used in conventional deblocking steps. Pyridine hydrochloride itself is an effective reagent for activating nucleoside methylphosphonoamidites and ribonucleoside phosphoramidites, as well as deoxyribonucleoside phosphoramidites, when high O/N selectivety is not needed.

Base Sequence↗
PubMed1991

Oxalyl-CPG: a labile support for synthesis of sensitive oligonucleotide derivatives.

A procedure is described for linking nucleosides covalently to controlled pore glass or cross-linked polystyrene supports by means of an oxalyl anchor. Though stable to triethylamine and diisopropylamine, the nucleoside-oxalyl link can be cleaved within a few minutes at room temperature with ammonium hydroxide in methanol. This new anchor can be used in automated synthesis of conventional oligonucleotides. The primary value, however, is that it enables one to employ solid support methodology to synthesize a variety of base-sensitive oligonucleotide derivatives, as illustrated here by synthesis of oligomers with base protecting groups intact and with methyl phosphotriester groups at the internucleoside links.

Base Sequence↗
PubMed1991

Mode of action of 5'-linked cholesteryl phosphorothioate oligodeoxynucleotides in inhibiting syncytia formation and infection by HIV-1 and HIV-2 in vitro.

A phosphorothioate homocytidine 10-mer containing a cholesteryl moiety covalently linked to the 5'-end (Chol-SdC10) inhibited syncytium formation in susceptible T cells induced by HIV-1 and HIV-2. The syncytium inhibition effect was minimal with unmodified cytidine homopolymer of the same net charge. Chol-SdC10 was shown to protect CEM cells against infection by cell-free HIV-1 particles without any apparent toxicity to the growth of CD4+ T cells. The DNA polymerase activity of the purified reverse transcriptase (RT) of HIV-1 was markedly inhibited by Chol-SdC10 but the effect on the RNase H activity of RT was minimal. Analysis of the kinetics of reverse transcriptase inhibition mediated by the drug revealed that the inhibition at a higher concentration was competitive with respect to template primer binding and noncompetitive at lower concentrations. Chol-SdC10 also partially blocked the binding of gp120 to CD4 in a solid-phase ELISA. These results confirm that the anti-HIV activity of phosphorothioate cytidine homopolymers increases markedly by covalent modification with the cholesteryl moiety at the 5'-end and demonstrates that the cytoprotective effect is manifested at multiple steps in the virus life cycle. These steps include inhibition of retroviral replication activity as well as the binding and fusion of HIV with CD4+ T cells.

Antiviral Agents↗
PubMed1991

Synthesis and properties of modified oligonucleotides.

Phosphorothioate oligonucleotide analogs conjugated to cholesteryl by a neutral, 6 atom linker are more effective inhibitors of HIV-1 in cell culture than the corresponding analogs conjugated via a phosphorothioate group. The antiviral activity correlates with the hydrophobic character of the oligonucleotide. Some new synthetic methodology is also discussed.

Antiviral Agents↗