Biomedical subjects

E T Kool

Publications and source records attributed to E T Kool.

42 records · Page 3Linked to original sources

PubMed1995

Relative stabilities of triple helices composed of combinations of DNA, RNA and 2'-O-methyl-RNA backbones: chimeric circular oligonucleotides as probes.

Described is a systematic study of the effects of varied backbone structure on the stabilities of pyr.pur.pyr triple helices. The effects were measured using six circular 34 base oligonucleotides containing DNA (D), RNA (R) and/or 2'-O-methyl-RNA (M) residues designed to bind a complementary single-stranded purine target strand by triple helix formation. Eighteen different backbone combinations were studied at pH 5.5 and 7.0 by optical melting experiments and the results compared with the stabilities of the corresponding Watson-Crick duplexes. When the target purine strand is DNA, all circles form pH-dependent triple helical complexes which are considerably stronger than the duplexes alone. When RNA is the target, five of the nine complexes studied are of the pH-dependent triplex type and the other four complexes are not significantly stronger than the corresponding duplexes. The results are useful in the design of the highest affinity ligands for single- and double-stranded DNAs and RNAs and also point out novel ways to engender DNA- or RNA-selective binding.

Base Sequence↗

PubMed1995

Origins of the large differences in stability of DNA and RNA helices: C-5 methyl and 2'-hydroxyl effects.

Recent studies have shown that there can be large differences in the stability of double and triple helical nucleic acid complexes, depending on whether RNA or DNA strands are involved. These differences have been attributed to structural differences in the sugar-phosphate backbone of these two polymers. However, since there are in fact two structural features which distinguish DNA from RNA (the 2'-hydroxyl and C-5 methyl groups), the stability differences may arise from either or both of these factors. We have separated effects of the 2'-hydroxyl and C-5 methyl groups by synthesizing nucleic acid strands which contain all possible combinations with and without these groups. Studies of the stabilities of double and triple helices involving these strands show that in fact the C-5 methyl group of thymine and the 2'-OH group of ribose have equally large effects on stability. The two effects vary with secondary structure and can be reinforcing or even opposing in their influence on stability. Three types of complexes are specifically examined: bimolecular pyrimidine.purine duplexes, termolecular pyrimidine.purine.pyrimidine triplexes, and bimolecular triplexes formed from circular pyrimidine oligonucleotides with purine target strands. It is found in general that the two types of substitutional effects are independent of one another and that C-5 methyl groups are in all cases stabilizing, while 2'-OH groups can be stabilizing or destabilizing, depending on the type of complex. In addition, studies with partially methylated duplexes lend evidence that the largest contribution to stabilization by the methyl group arises from increased base stacking ability rather than from a favorable hydrophobic methyl-methyl contact.(ABSTRACT TRUNCATED AT 250 WORDS)

Amides↗

PubMed1994

Circular RNA oligonucleotides. Synthesis, nucleic acid binding properties, and a comparison with circular DNAs.

We report the synthesis and nucleic acid binding properties of two cyclic RNA oligonucleotides designed to bind single-stranded nucleic acids by pyr.pur.pyr-type triple helix formation. The circular RNAs are 34 nucleotides in size and were cyclized using a template-directed nonenzymatic ligation. To ensure isomeric 3'-5' purity in the ligation reaction, one nucleotide at the ligation site is a 2'-deoxyribose. One circle (1) is complementary to the sequence 5'-A12, and the second (2) is complementary to 5'-AAGAAAGAAAAG. Results of thermal denaturation experiments and mixing studies show that both circles bind complementary single-stranded DNA or RNA substrates by triple helix formation, in which two domains in a pyrimidine-rich circle sandwich a central purine-rich substrate. The affinities of these circles with their purine complements are much higher than the affinities of either the linear precursors or simple Watson-Crick DNA complements. For example, circle 1 binds rA12 (pH 7.0, 10 mM MgCl2, 100 mM NaCl) with a Tm of 48 degrees C and a Kd (37 degrees C) of 4.1 x 10(-9) M, while the linear precursor of the circle binds with a Tm of 34 degrees C and a Kd of 1.2 x 10(-6) M. The complexes of circle 2 are pH-dependent, as expected for triple helical complexes involving C(+)G.C triads, and mixing plots for both circles reveal one-to-one stoichiometry of binding either to RNA or DNA substrates. Comparison of circular RNAs with previously synthesized circular DNA oligonucleotides of the same sequence reveals similar behavior in the binding of DNA, but strikingly different behavior in the binding of RNA. The cyclic DNAs show high DNA-binding selectivity, giving relatively weaker duplex-type binding with complementary RNAs. The relative order of thermodynamic stability for the four types of triplex studied here is found to be DDD >> RRR > RDR >> DRD. The results are discussed in the context of recent reports of strong triplex dependence on RNA versus DNA backbones. Triplex-forming circular RNAs represent a novel and potentially useful strategy for high-affinity binding of RNA.

Base Sequence↗

PubMed1994

Stabilities of nucleotide loops bridging the pyrimidine strands in DNA pyrimidine.purine.pyrimidine triplexes: special stability of the CTTTG loop.

Recent studies of DNA hairpin loops have shown considerable dependence of the stability on the sequence of the loop [Senior, M., Jones, R. A., & Breslauer, K. J. (1988a) Proc. Natl. Acad. Sci. U.S.A. 85, 6242-6246; Xodo, L. E., Manzini, G., Quadrifoglio, F., van der Marel, G., & van Boom, J. H. (1989) Biochimie 71, 793-803; Hirao, I., Nishimura, Y., Tagawa, Y., Watanabe, K., & Miura, K. (1992) Nucleic Acids Res. 20, 3891-3896]. Analogous studies have not, until now, been carried out with loops in triple helices. We report the results from experiments in which we examine the relative stabilities of pentanucleotide loops that bridge between the pyrimidine strands in DNA pyr.pur.pyr triple helices. There are two types of loops that are defined by the relative orientation of the purine strand: a 5'-loop and a 3'-loop. The sequences examined in this study are the bimolecular triplexes formed between 5'-dTTCTTTTCL1TTTL5CTTTTCTT (loop nucleotides are underlined, and L1 and L5 represent varied nucleotides) and the two purine strands, 5'-dAAGAAAAG-3' and 5'-dGAAAAGAA-3'. The first and last nucleotides in the loop are varied, since stacking interactions may be strongest at these positions [Senior et al., 1988a; Senior, M., Jones, R. A., & Breslauer, K. J. (1988b) Biochemistry 27, 3879-3885], and we examine 14 sequence combinations for each loop type. Thermal denaturation studies carried out at pH 7.0 indicate considerable variation in the stabilities of these loops.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1994

Base pairing and steric interactions between pyrimidine strand bridging loops and the purine strand in DNA pyrimidine.purine.pyrimidine triplexes.

Bimolecular triple-helical DNA complexes recently have found use in a new strategy for the recognition of single-stranded nucleic acids, in which circular (Kool, 1991; Prakash & Kool, 1992) or hairpin-shaped (Giovannangeli et al., 1991; D'Souza & Kool, 1992) oligonucleotides bind these single strands by triplex formation. Bimolecular triplexes may also be formed in vivo as H-DNA, where this structure may potentially play a role in gene expression and recombination (Belotserkovskii et al., 1990; Hanvey et al., 1989; Shimizu et al., 1989). In all of these complexes, the central strand of the triplex must pass beyond the loop that bridges the outer two strands, and models and preliminary experiments have indicated that there may be important interactions between this central strand and the loop (Prakash & Kool, 1992). We now report thermal denaturation studies carried out specifically to investigate these interactions in detail, using as a model the 5'-loop and 3'-loop complexes formed between 14 pyrimidine oligodeoxynucleotides having the sequence 5'-dTTCTTTTCL1TTTL5CTTTTCTT, where L1 and L5 represent varied nucleotides in the loop (which is underlined), and eight target strands having the sequence 5'-dCCCCFAAGAAAAG-3' or 5'-dGAAAAGAAFCCCCC-3', where F is a varied nucleotide flanking the triplex in the central strand. Results correlated from 64 different sequence combinations show that there is wide variation in the stabilities of the complexes, indicating specific and substantial interactions between the nucleotides at the L1, F, and L5 positions. Melting temperatures at pH 7.0 range from 17.0 degrees C to 34.6 degrees C, and free energies (37 degrees C) range from -3.2 to -7.8 kcal mol-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Composition↗

PubMed1992

Strong binding of single-stranded DNA by stem-loop oligonucleotides.

We report that oligodeoxynucleotides which form stem-loop hairpin structures and which have pyrimidine-rich loops can form strong complexes with complementary single-stranded DNA sequences. Stem-loop oligonucleotides were constructed with a 25-nt T-rich loop and with variable Watson-Crick stems. The complexes of these oligomers with the sequence dA8 were studied by thermal denaturation. Evidence is presented that the complexes are one-to-one, bimolecular complexes in which the pyrimidine loop bases comprise the outer strands in a pyr.pur.pyr triplex, in effect chelating the purine strand in the center of the loop. Melting temperatures for the loop complexes are shown to be up to 29 degrees C higher than Watson-Crick duplex of the same length. It is shown that the presence of a stem increases stability of the triplex relative to an analogous oligomer without a stem. The effect of stem length on the stability of such a complex is examined. Such hairpin oligomers represent a new approach to the sequence-specific binding of single-stranded RNA and DNA. In addition, the finding raises the possibility that such a complex may exist in natural RNA folded sequences.

Base Sequence↗