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S M Freier

Publications and source records attributed to S M Freier.

At least 19 recordsLinked to original sources
PubMed1995

Relative thermodynamic stability of DNA, RNA, and DNA:RNA hybrid duplexes: relationship with base composition and structure.

Fourteen oligonucleotides 8-21 nucleotides in length and their complements were synthesized as DNA and RNA. For each sequence, four kinds of duplexes, DNA:DNA, RNA:RNA, DNA:RNA, and RNA:DNA, were prepared. Twelve sequences had A.T/U content varying from 25 to 80% and dPy content in the DNA strands varying from 0 to 100%. Thermodynamic stabilities of four duplexes for each sequence were determined in solution containing 100 mM Na+, 10 mM phosphate, and 0.1 mM EDTA, pH 7.1. CD spectra and electrophoretic mobility on native polyacrylamide gel were measured for most duplexes. Quantitative correlations of hybrid stability both with deoxypyrimidine content and, at fixed dPy content, with the fraction of A.T/U in duplexes were found. We also demonstrated that hybrids with 70-80% deoxypyrimidine DNA strand and a high or moderate A.T/U fraction displayed the highest relative stability compared to their RNA counterparts. Relationships of relative intensities of CD bands at 210 nm and relative electrophoretic mobilities of hybrids with relative hybrid stability suggested that hybrid conformation varies continuously between A- and B-form and is the decisive factor in relative hybrid stability.

Base Sequence↗
PubMed1995

Inhibition of NF-kappa B specific transcriptional activation by PNA strand invasion.

Peptide nucleic acid (PNA) strand invasion offers an attractive alternative to DNA oligonucleotide directed triplex formation as a potential tool for gene inhibition. Peptide nucleic acid has been shown to interact with duplex DNA in a process which involves strand invasion of the duplex and binding of one of the DNA strands with two PNA oligomers. By blocking the interaction of a transcription factor with 5' regulatory sequences, PNA might specifically down-regulate gene activity. Here we demonstrate that PNA is capable of specifically blocking interaction of the transcription factor NF-kappa B with the IL2-R alpha NF kappa-B binding site in vitro. We further demonstrate that this interaction is sufficient to prevent transcriptional transactivation both in vitro and when transfected into cells in culture.

Base Sequence↗
PubMed1995

Characterization of fully 2'-modified oligoribonucleotide hetero- and homoduplex hybridization and nuclease sensitivity.

The nuclease stability and melting temperatures (Tm) were compared for fully modified oligoribonucleotide sequences containing 2'-fluoro, 2'-O-methyl, 2'-O-propyl and 2'-O-pentyl nucleotides. Duplexes formed between 2' modified oligoribonucleotides and RNA have typical A-form geometry as observed by circular dichroism spectroscopy. Modifications, with the exception of 2'-O-pentyl, were observed to increase the Tm of duplexes formed with complementary RNA. Modified homoduplexes showed significantly higher Tms, with the following Tm order: 2'-fluoro:2'fluoro > 2'-O-propyl:2'-O-propyl > 2'-O-methyl:2'-O- methyl > RNA:RNA > DNA:DNA. The nuclease stability of 2'-modified oligoribonucleotides was examined using snake venom phosphodiesterase (SVPD) and nuclease S1. The stability imparted by 2' modifications was observed to correlate with the size of the modification. An additional level of nuclease stability was present in oligoribonucleotides having the potential for forming secondary structure, but only for 2' modified oligoribonucleotides and not for 2'-deoxy oligoribonucleotides.

Base Sequence↗
PubMed1995

Deconvolution of combinatorial libraries for drug discovery: a model system.

Iterative synthesis and screening strategies have recently been used to identify unique active molecules from complex synthetic combinatorial libraries. These techniques have many advantages over traditional screening methods, including the potential to screen large numbers of compounds to identify an active molecule while avoiding analytical separations and structural determination of unknown compounds. It is not clear, however, whether these techniques identify the most active molecular species in the mixtures and, if so, how often. Two key factors which may affect success of the selection process are the presence of many active compounds in the library with a range of activities and the chosen order of unrandomization. The importance of these factors has not been previously studied. Moreover, the impact of experimental errors in determination of subset activities or in randomization during library synthesis is not known. We describe here a model system based on oligonucleotide hybridization that addresses these questions using computer simulations. The results suggested that, within achievable experimental and library synthesis error, iterative deconvolution methods generally find either the best molecule or one with activity very close to the best. The presence of many active compounds in a library influenced the profile of subset activities, but did not preclude selection of a molecule with near optimal activity.

Base Sequence↗
PubMed1993

PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules.

DNA analogues are currently being intensely investigated owing to their potential as gene-targeted drugs. Furthermore, their properties and interaction with DNA and RNA could provide a better understanding of the structural features of natural DNA that determine its unique chemical, biological and genetic properties. We recently designed a DNA analogue, PNA, in which the backbone is structurally homomorphous with the deoxyribose backbone and consists of N-(2-aminoethyl)glycine units to which the nucleobases are attached. We showed that PNA oligomers containing solely thymine and cytosine can hybridize to complementary oligonucleotides, presumably by forming Watson-Crick-Hoogsteen (PNA)2-DNA triplexes, which are much more stable than the corresponding DNA-DNA duplexes, and bind to double-stranded DNA by strand displacement. We report here that PNA containing all four natural nucleobases hybridizes to complementary oligonucleotides obeying the Watson-Crick base-pairing rules, and thus is a true DNA mimic in terms of base-pair recognition.

Base Sequence↗
PubMed1993

Oligodeoxynucleotides containing 2'-O-modified adenosine: synthesis and effects on stability of DNA:RNA duplexes.

Hybridization thermodynamics were compared for oligonucleotide sequences containing 2'-fluoro dA, 2'-O-methyl A, 2'-O-ethyl A, 2'-O-propyl A, 2'-O-butyl A, 2'-O-pentyl A, 2'-O-nonyl A, 2'-O-allyl A, and 2'-O-benzyl A in place of deoxyadenosine. Although the effect of 2'-modified adenosine on duplex stability is sequence dependent, a clear trend is apparent. For six sequences containing a few 2'-modified adenosines in a background of unmodified deoxynucleotides, the average delta TM per substitution ranged from +1.3 degrees C for 2'-fluoro dA to -2.0 degrees C for 2'-O-nonyl A. For the 2'-O-alkyl series, the average delta TM per substitution correlates well with size of the substituent; the order of stability is 2'-O-methyl A > 2'-O-ethyl A > 2'-O-propyl A > 2'-O-butyl A > 2'-O-pentyl A > 2'-O-nonyl A. This correlation also extends to 2'-fluoro dA, 2'-O-allyl A, and 2'-O-benzyl A if chain length is measured by number of carbon atoms. When examined in the background of 2'-O-methyl ribonucleotides, all 2'-modified adenosines with a substituent no larger than 2'-O-pentyl stabilized the duplex nearly 2 degrees C per substitution compared to unmodified dA. These thermodynamic results and CD spectra of modified and unmodified hybrids support a model of DNA:RNA hybrids in which the geometry is between that of B-form and A-form.

Adenosine↗
PubMed1992

Implication of RNA structure on antisense oligonucleotide hybridization kinetics.

A 47-nucleotide transcript of the activated Ha-ras gene was prepared and determined, by enzymatic structure mapping, to form a stable hairpin structure. Six antisense decaribonucleotides were designed, and association constants (Ka) for the hairpin- and length-matched complements were measured. Two of the antisense oligonucleotides targeted to the loop had nearly equal affinity for the transcript compared to the complement. The others, including one oligonucleotide complementary to the 3' side of the single-stranded loop, bound 10(5)-10(6)-fold less tightly to the transcript than to the short complement. We propose the difference in affinity is due to the target structure, both the secondary structure of the stem and the structure in the loop. Measurement of the bimolecular association rate constant, k1, and the dissociation rate constant, k-1, for these oligonucleotides indicates the observed relationship between affinity and structure is primarily due to k1.

Base Sequence↗
PubMed1992

Selective inhibition of mutant Ha-ras mRNA expression by antisense oligonucleotides.

A biological reporter gene assay was employed to determine the crucial parameters for maximizing selective targeting of a Ha-ras codon 12 point mutation (G----T) using phosphorothioate antisense oligonucleotides. We have tested a series of oligonucleotides ranging in length between 5 and 25 bases, each centered around the codon 12 point mutation. Our results indicate that selective targeting of this point mutation can be achieved with phosphorothioate antisense oligonucleotides, but this selectivity is critically dependent upon oligonucleotide length and concentration. The maximum selectivity observed in antisense experiments, 5-fold for a 17-base oligonucleotide, was closely predicted by a simple thermodynamic model that relates the fraction of mutant to wild type target bound as a function of oligonucleotide concentration and affinity. These results suggest thermodynamic analysis of oligonucleotide/target interactions is useful in predicting the specificity that can be achieved by an antisense oligonucleotide targeted to a single base point mutation.

Amino Acid Sequence↗
PubMed1992

Pseudo--half-knot formation with RNA.

A pseudo--half-knot can be formed by binding an oligonucleotide asymmetrically to an RNA hairpin loop. This binding motif was used to target the human immunodeficiency virus TAR element, an important viral RNA structure that is the receptor for Tat, the major viral transactivator protein. Oligonucleotides complementary to different halves of the TAR structure bound with greater affinity than molecules designed to bind symmetrically around the hairpin. The pseudo--half-knot--forming oligonucleotides altered the TAR structure so that specific recognition and binding of a Tat-derived peptide was disrupted. This general binding motif may be used to disrupt the structure of regulatory RNA hairpins.

Base Sequence↗
PubMed1991

Effects of phosphorothioate capping on antisense oligonucleotide stability, hybridization and antiviral efficacy versus herpes simplex virus infection.

Efforts have been made to improve the biological stability of phosphodiester (PO) oligonucleotides by the addition of various modifications to either the 3', 5' or both the 3' and 5' ends of an oligonucleotide. ISIS 1080, a phosphorothioate (PS) 21-mer oligonucleotide complementary to the internal AUG codon of UL13 mRNA in HSV-1, reduces the infectious yield of HSV-1 in HeLa cells to 9.0% +/- 11%. PO analogs of ISIS 1080 containing three PS linkages placed on the 3' (ISIS 1365), 5' (ISIS 1370), both the 3' and 5' (ISIS 1364) ends or with four linkages in the middle (ISIS 1400) demonstrated reduced antiviral efficacy compared to fully PS ISIS 1080. Thermal denaturation profiles demonstrated that these oligonucleotides hybridized to complementary DNA or RNA with equivalent binding affinities. All were able to support E. coli RNAse H cleavage of the HSV mRNA to which they were targeted. The stability of the congeners in cell culture medium containing 10% fetal calf serum (FCS), HeLa cytosolic extract, HeLa nuclear extract and in intact HeLa cells revealed that ISIS 1080 was most resistant to nucleolytic digestion through 48 hours. Partial PS oligonucleotides exhibited increased degradation compared to the fully thioated oligonucleotide by exonuclease activity in FCS and endonuclease activity in cell extracts or intact cells. Thus, the reduced efficacy of partial compared to fully PS oligonucleotides against HSV-1 in HeLa cells may result from increased degradation of the mixed PO/PS oligonucleotides.

Antiviral Agents↗
PubMed1991

Antisense oligonucleotides inhibit intercellular adhesion molecule 1 expression by two distinct mechanisms.

Intercellular adhesion molecule 1 (ICAM-1) is a glycoprotein expressed on the surface of both hemopoietic and nonhemopoietic cells that mediates, in part, the emigration of leukocytes out of the vasculature. Expression of ICAM-1 on the surface of human umbilical vein endothelial cells and a human lung carcinoma cell line (A549) was increased by interleukin-1 beta, tumor necrosis factor alpha, and interferon gamma in a concentration-dependent manner. Phosphorothioate antisense oligonucleotides designed to hybridize to 10 target sites on the human ICAM-1 mRNA were tested for inhibition of ICAM-1 expression in both cell lines by an ICAM-1 enzyme-linked immunosorbent assay. Based upon potency and unique mRNA target sites, two oligonucleotides were studied in greater detail: ISIS 1570, which targeted the AUG translation initiation codon, and ISIS 1939, which targeted specific sequences in the 3'-untranslated region of the mRNA. Both oligonucleotides specifically inhibit expression of ICAM-1 as analyzed by immunoprecipitation of 35S-labeled proteins. Treatment of cells with ISIS 1939 promoted a reduction in ICAM-1 mRNA, whereas ISIS 1570 did not change the level of ICAM-1 mRNA, suggesting that the two oligonucleotides may be inhibiting ICAM-1 expression by two different mechanisms. The activity of both oligonucleotides was blocked by hybridization of the oligonucleotide to its complementary sense strand prior to addition to the cells. Neither ISIS 1570 nor ISIS 1939 changed the transcriptional rate of the ICAM-1 gene, demonstrating that both oligonucleotides were working through a post-transcriptional mechanism. 2'-O-Methyl phosphorothioate analogs, which do not support RNase H-mediated cleavage of target mRNA, were used to determine if the active antisense oligonucleotides inhibited ICAM-1 expression by an RNase H-dependent mechanism. The 2'-O-methyl phosphorothioate analog of ISIS 1939 did not significantly reduce interleukin-1 beta-induced ICAM-1 expression, whereas the 2'-O-methyl phosphorothioate analog of ISIS 1570 did inhibit ICAM-1 expression, suggesting that the reduction of ICAM-1 mRNA following treatment with ISIS 1939 was due, in part, to RNase H-mediated hydrolysis. Adherence of HL-60 cells to human umbilical vein cell monolayers was inhibited by ISIS 1570 and ISIS 1939, demonstrating that the reduced levels of ICAM-1 impact on ICAM-1-associated function.

Amino Acid Sequence↗
PubMed1989

Laser temperature-jump, spectroscopic, and thermodynamic study of salt effects on duplex formation by dGCATGC.

Salt effects on duplex formation by dGCATGC have been studied with spectroscopic, thermodynamic, and kinetic methods. Circular dichroism spectra indicate different salt conditions have little effect on the structures of the duplex and single strand. NMR chemical shifts indicate the structure of the duplex in 1 M NaCl is similar to that of the B-form determined previously in 0.5 M KCl [Nilges, M., Clore, G. M., Gronenborn, A. M., Brunger, A. T., Karplus, M., & Nilsson, L. (1987) Biochemistry 26, 3718-3733]. Optical melting experiments indicate the effect of Na+ concentration on melting temperature is similar to that expected for a polynucleotide with the same GC content. Laser temperature-jump experiments indicate the effect of Na+ concentration on the rate of duplex formation is much less than is observed for polynucleotides. The observations are consistent with expectations based on a counterion condensation model. This is surprising for a duplex with only 10 phosphates.

Circular Dichroism↗
PubMed1987

Improved parameters for prediction of RNA structure.

Thermodynamic studies of oligoribonucleotides are providing parameters and insights for the fundamental interactions that determine RNA structure. These results can be used to predict the secondary structure of RNA from its sequence. Comparisons of predicted structures with those deduced from phylogenetic data indicate a modest success rate that is improving as more parameters are determined experimentally. Two major fundamental interactions in RNA are stacking and hydrogen bonding. Both contribute similar increments to free-energy changes for associations of oligoribonucleotides. Thus, parameters for stacking and hydrogen bonding will likely be important for predicting the three-dimensional structures of RNAs and for interpreting RNA-RNA associations. Both applications should be important for providing a full understanding of catalysis by RNA.

Animals↗
PubMed1986

Polymer-supported RNA synthesis and its application to test the nearest-neighbor model for duplex stability.

A solid-phase method using a phosphoramidite approach is described for synthesis of oligoribonucleotides. The method was used to synthesize pairs of oligomers with identical nearest neighbors but different sequences. Comparison of thermodynamic parameters for these pairs provides a test of the nearest-neighbor hypothesis for prediction of helix stability. In general, pairs of sequences with identical nearest neighbors have enthalpy and entropy changes for helix formation that differ by 8% on average, delta Go37 that differ by 6% on average, and melting temperatures within 0-5 degrees C of each other. These limits are typical of the accuracy that should be expected from nearest-neighbor predictions of RNA helix stability. UCAUGA and UGAUCA have the same nearest neighbors but melting temperatures that differ by 7 degrees C. This suggests some sequences will not be approximated well by the nearest-neighbor model.

Base Sequence↗
PubMed1986

Energetics of internal GU mismatches in ribooligonucleotide helixes.

Thermodynamic parameters of helix formation were measured spectroscopically for 16 oligoribonucleotides containing either internal GU mismatches or the corresponding AU pairs. Internal GU mismatches stabilize each helix, but not as much as the corresponding AU pairs. The differences in the enthalpy and entropy changes of helix formation associated with replacing AU pairs with GU mismatches are less than previously realized. At both 25 and 37 degrees C, the decrease in helix stability associated with replacing an AU with a GU is also less than thought previously. Approximations are suggested for predicting the effects of GU mismatches on helix stability.

Base Composition↗
PubMed1986

Free energy contributions of G.U and other terminal mismatches to helix stability.

Thermodynamic parameters of helix formation were measured spectroscopically for seven hexaribonucleotides containing a GC tetramer core and G.U or other terminal mismatches. The free energies of helix formation are compared with those for the tetramer core alone and with those for the hexamer with six Watson-Crick base pairs. In 1 M NaCl, at 37 degrees C, the free energy of a terminal G.U mismatch is about equal to that of the corresponding A.U pair. Although other terminal mismatches studied add between -1.0 and -1.6 kcal/mol to delta G0 37 for helix formation, all are less stable than the corresponding Watson-Crick pairs. Comparisons of the stability increments for terminal G.U mismatches and G.C pairs suggest when stacking is weak the additional hydrogen bond in the G.C pair adds roughly -1 kcal/mol to the favorable free energy of duplex formation.

Base Composition↗
PubMed1986

Stability of XGCGCp, GCGCYp, and XGCGCYp helixes: an empirical estimate of the energetics of hydrogen bonds in nucleic acids.

The stabilizing effects of dangling ends and terminal base pairs on the core helix GCGC are reported. Enthalpy and entropy changes of helix formation were measured spectrophotometrically for AGCGCU, UGCGCA, GGCGCCp, CGCGCGp, and the corresponding pentamers XGCGCp and GCGCYp containing the GCGC core plus a dangling end. Each 5' dangling end increases helix stability at 37 degrees C roughly 0.2 kcal/mol and each 3' end from 0.8 to 1.7 kcal/mol. The free energy increments for dangling ends on GCGC are similar to the corresponding increments reported for the GGCC core [Freier, S. M., Alkema, D., Sinclair, A., Neilson, T., & Turner, D. H. (1985) Biochemistry 24, 4533-4539], indicating a nearest-neighbor model is adequate for prediction of stabilization due to dangling ends. Nearest-neighbor parameters for prediction of the free energy effects of adding dangling ends and terminal base pairs next to G.C pairs are presented. Comparison of these free energy changes is used to partition the free energy of base pair formation into contributions of "stacking" and "pairing". If pairing contributions are due to hydrogen bonding, the results suggest stacking and hydrogen bonding make roughly comparable favorable contributions to the stability of a terminal base pair. The free energy increment associated with forming a hydrogen bond is estimated to be -1 kcal/mol of hydrogen bond.

Calorimetry↗