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Biomedical subjects

E T Kool

Publications and source records attributed to E T Kool.

At least 37 records · Page 2Linked to original sources
PubMed1998

Probing DNA sequences in solution with a monomer-excimer fluorescence color change.

The use of a simple fluorescent nucleoside analogue in detection of point mutations by hybridization in solution is described. Pyrene is placed at 3' and 5' ends of a pair of oligodeoxynucleotide probes via a phosphoramidite derivative of deoxyribose with this fluorophore attached at the 1' position, replacing a DNA base. Adjacent binding of dual probes containing this fluorophore to a complementary target sequence results in a pronounced spectral change from blue pyrene monomer emission (lambdamax= 381 398 nm) to green-white excimer emission (lambdamax= 490 nm). Optimization of the relative binding positions of the two probes shows that the greatest spectral change occurs when they bind with partial end overlap. In optimum orientation, the monomer emission band for the probes decreases intensity by as much as a factor of seven and the excimer band increases up to 40-fold on binding a complementary target. Application to the detection of a single-base point mutation in solution is described.

Base Sequence↗
PubMed1998

Chemical and enzymatic properties of bridging 5'-S-phosphorothioester linkages in DNA.

We describe physicochemical and enzymatic properties of 5' bridging phosphorothioester linkages at specific sites in DNA oligonucleotides. The susceptibility to hydrolysis at various pH values is examined and no measurable hydrolysis is observed at pH 5-9 after 4 days at 25 degrees C. The abilities of three 3'- and 5'-exonuclease enzymes to hydrolyze the DNA past this linkage are examined and it is found that the linkage causes significant pauses at the sulfur linkage for T4 DNA polymerase and calf spleen phosphodiesterase, but not for snake venom phosphodiesterase. Restriction endonuclease (Nsi I) cleavage is also attempted at a 5'-thioester junction and strong resistance to cleavage is observed. Also tested is the ability of polymerase enzymes to utilize templates containing single 5'-S-thioester linkages; both Klenow DNA polymerase and T7 RNA polymerase are found to synthesize complementary strands successfully without any apparent pause at the sulfur linkage. Finally, the thermal stabilities of duplexes containing such linkages are measured; results show that T m values are lowered by a small amount (2 degrees C) when one or two thioester linkages are present in an otherwise unmodified duplex. The chemical stability and surprisingly small perturbation by the 5' bridging sulfur make it a good candidate as a physical and mechanistic probe for specific protein or metal interactions involving this position in DNA.

Animals↗
PubMed1998

Generation of circular RNAs and trans-cleaving catalytic RNAs by rolling transcription of circular DNA oligonucleotides encoding hairpin ribozymes.

A simple new strategy for the in vitro synthesis of circular RNAs and hairpin ribozymes is described. Circular single-strand DNA oligonucleotides 67-79 nt in length are constructed to encode both hairpin ribozyme sequences and ribozyme-cleavable sequences. In vitro transcription of these small circles by Escherichia coli RNA polymerase produces long repeating RNAs by a rolling circle mechanism. These repetitive RNAsundergo self-processing, eventually yielding unit length circular and linear RNAs as the chief products. The transcription is efficient despite the absence of promoter sequences, with RNA being produced in up to 400 times the amount of DNA circle used. It is shown that the linear monomeric hairpin ribozymes are active in cleaving RNA targets in trans , including one from HIV-1. Several new findings are established: (i) that rolling circle transcription can be extended to the synthesis of catalytic RNAs outside the hammerhead ribozyme motif; (ii) that rolling circle transcription is potentially a very simple and useful strategy for the generation of circular RNAs in preparative amounts; and (iii) that self-processed hairpin ribozymes can be catalytically active in trans despite the presence of self-binding domains.

Base Sequence↗
PubMed1998

Replication of non-hydrogen bonded bases by DNA polymerases: a mechanism for steric matching.

Recent experiments have presented evidence that Watson-Crick hydrogen bonds in a base pair are not absolute requirements for efficient synthesis of that pair by DNA polymerase enzymes. Here we examine quantitative steady-state kinetic data from several published studies involving poorly hydrogen-bonding DNA base analogues and adducts, and analyze the results in terms of solvation, hydrogen bonding, and steric effects. We propose a mechanism that can explain the surprising lack of hydrogen-bonding requirement accompanied by significant selectivity in pairing. This hypothesis makes use of steric matching, enforced both by the tightly confined polymerase active site and by the DNA backbone, as a chief factor determining nucleotide selection during DNA synthesis. The results also suggest that hydrogen bonds from bases to water (solvation) may be important in increasing the effective size of DNA bases, which may help prevent misinsertion of small bases opposite each other.

DNA Replication↗
PubMed1998

Triplex-directed self-assembly of an artificial sliding clamp on duplex DNA.

BACKGROUND: Circular triplex-forming oligonucleotides (CTFOs) have previously been shown to bind tightly to short single-stranded homopurine DNAs in a sequence-specific manner. In view of the importance of double-stranded DNA as a target in the development of gene-specific therapeutic and diagnostic agents, we have investigated the binding of CTFOs to double-helical DNA. RESULTS: DNA-binding experiments show that a CTFO can recognize its homopurine target when the target is embedded in a long duplex. Unlike their linear counterparts, CTFOs bind the double helix in two topologically distinct forms. The more stable of the two complexes is found to be a pseudorotaxane, having the same topology as the sliding clamp protein subunits associated with some DNA and RNA polymerases. CONCLUSIONS: Circular triplex-forming oligonucleotides have been shown to bind the DNA double helix in a topological manner which is unprecedented among synthetic ligands. This novel binding motif allows a synthetic CTFO to be irreversibly locked onto a circular double-stranded DNA target without covalently modifying the target.

Binding Sites↗
PubMed1998

Efficient replication between non-hydrogen-bonded nucleoside shape analogs.

DNA polymerase enzymes make an error only once per 10(4)-10(5) initial nucleotide insertions during DNA replication. Most currently held models of this high fidelity cite the hydrogen bonds between complementary pyrimidines and purines as a critical controlling factor. Testing this has been difficult, however, since standard molecular strategies for blocking or removing polar hydrogen-bonding groups cause changes to size and shape as well as hydrogen bonding ability. One answer to this problem is the use of nonpolar molecules that mimic the shape of natural DNA bases. Here we show that a non-hydrogen-bonding shape mimic for adenine is replicated efficiently and selectively against a nonpolar shape mimic for thymine. The results establish that hydrogen bonds in a base pair are not absolutely required for efficient nucleotide insertion. This adds support to the idea that shape complementarity may play as important a role in replication as base-base hydrogen bonds.

Adenosine Triphosphate↗
PubMed1998

Solution structure of a DNA duplex containing a replicable difluorotoluene-adenine pair.

A nonpolar aromatic nucleoside derivative based on 2,4-difluorotoluene (F), a non-hydrogen bonding shape analog of thymidine, was recently shown to be replicated against adenine with high efficiency and fidelity. This led to the suggestion that geometric matching, potentially even in the absence of hydrogen bonding between bases in a pair, may be sufficient to direct nucleotide selection during replication. We have examined the solution structure of the F-A pair in the context of a 12 base pair DNA duplex. We find that, despite the destabilization caused by this analog, the F-A pair very closely resembles that of a T x A pair in the same context. This lends support to the importance of shape matching in replication.

Adenine↗
PubMed1997

A thymidine triphosphate shape analog lacking Watson-Crick pairing ability is replicated with high sequence selectivity.

Compound 1 (F), a nonpolar nucleoside analog that is isosteric with thymidine, has been proposed as a probe for the importance of hydrogen bonds in biological systems. Consistent with its lack of strong H-bond donors or acceptors, F is shown here by thermal denaturation studies to pair very poorly and with no significant selectivity among natural bases in DNA oligonucleotides. We report the synthesis of the 5'-triphosphate derivative of 1 and the study of its ability to be inserted into replicating DNA strands by the Klenow fragment (KF, exo- mutant) of Escherichia coli DNA polymerase I. We find that this nucleotide derivative (dFTP) is a surprisingly good substrate for KF; steady-state measurements indicate it is inserted into a template opposite adenine with efficiency (Vmax/Km) only 40-fold lower than dTTP. Moreover, it is inserted opposite A (relative to C, G, or T) with selectivity nearly as high as that observed for dTTP. Elongation of the strand past F in an F-A pair is associated with a brief pause, whereas that beyond A in the inverted A-F pair is not. Combined with data from studies with F in the template strand, the results show that KF can efficiently replicate a base pair (A-F/F-A) that is inherently very unstable, and the replication occurs with very high fidelity despite a lack of inherent base-pairing selectivity. The results suggest that hydrogen bonds may be less important in the fidelity of replication than commonly believed and that nucleotide/template shape complementarity may play a more important role than previously believed.

Base Composition↗
PubMed1997

Escherichia coli RNA polymerase activity observed using atomic force microscopy.

Fluid tapping-mode atomic force microscopy (AFM) was used to observe Escherichia coli RNA polymerase (RNAP) transcribing two different linear double-stranded (ds) DNA templates. The transcription process was detected by observing the translocation of the DNA template by RNAP on addition of ribonucleoside 5'-triphosphates (NTPs) in sequential AFM images. Stalled ternary complexes of RNAP, dsDNA and nascent RNA were adsorbed onto a mica surface and imaged under continuously flowing buffer. On introduction of all four NTPs, we observed some DNA molecules being pulled through the RNAP, some dissociating from the RNAP and others which did not move relative to the RNAP. The transcription rates were observed to be approximately 0.5-2 bases/s at our NTP concentrations, approximately 5 microM. The RNA transcripts were not unambiguously imaged in fluid. However, in experiments using a small single-stranded (ss) circular DNA template, known as a rolling circle, transcripts up to 1 or 2 microns long could be observed with tapping mode AFM once the samples were dried and imaged in air. This confirmed our observations of the transcriptional activity of RNAP adsorbed onto mica. This work illustrates that the development of tapping-mode in fluid has made it possible to use AFM to follow biological processes at the molecular level and get new insights about the variability of activity of individual molecules bound to a surface.

Aluminum Silicates↗
PubMed1997

Recognition of RNA by triplex formation: divergent effects of pyrimidine C-5 methylation.

In DNA triple helices, methylation at C-5 of thymine or cytosine is reported to have similar stabilizing effects for both bases. Here we show, however, that methylation of the same positions in RNA triplexes has distinctly different effects than in DNA. We have previously described the use of circular triplex-forming RNA oligonucleotides to recognize RNA sequences. Here it is shown that addition of C-5 methyl groups to uracils in these compounds very significantly increases not only affinity but also sequence selectivity in binding a purine-rich RNA target, as measured by thermal denaturation with various target RNAs. Surprisingly, however, addition of C-5 methyl groups to cytosines actually decreases affinity in binding RNA, while the same substitution in DNA is thermally stabilizing. Possible sources of this divergent behavior are discussed. A synthesis of 5-methylcytidine ribonucleoside 2'-O-silyl-3'-O-phosphoramidite is also described.

Base Sequence↗
PubMed1997

Bi-stranded, multisite replication of a base pair between difluorotoluene and adenine: confirmation by 'inverse' sequencing.

BACKGROUND: The nonpolar nucleoside of difluorotoluene (F) was previously found to behave similarly to thymidine in single-site deoxynucleoside triphosphate (dNTP) insertion experiments with the Klenow fragment (KF) of DNA polymerase I. Further study was needed, first to see whether F-A base pairs could be replicated in more than one sequence context; second to investigate whether specific base pair replication occurs in the presence of four dNTPs; and third to confirm the presence of F in a replicated DNA strand. RESULTS: A primer bound to a template strand containing eight F residues was extended by KF using the four natural dNTPs at 20 microM. Similarly, the complement (containing eight adenines) was extended using dATP, dGTP, dCTP and dFTP. Comparison of the new strands to authentic strands using standard and 'inverse' chemical sequencing showed identical composition within +/- 5%. CONCLUSIONS: The results confirm that F in a template strand encodes the insertion of dATP and that adenine in a template encodes the insertion of dFTP with good specificity in at least six different nearest neighbor contexts. The results confirm that analog F behaves similarly to thymidine despite its poor hydrogen-bonding ability.

Adenine↗
PubMed1997

Generation of catalytic RNAs by rolling transcription of synthetic DNA nanocircles.

Small catalytic RNAs are commonly produced either by transcription of promoter-driven linear DNA templates or by stepwise chemical synthesis on solid supports. We describe a different approach, in which very small chemically synthesized circular DNAs serve as efficient templates for generation of catalytic RNAs in vitro. The circles are 83 nucleotides in size, are single stranded, and contain no canonical RNA polymerase promoters. Despite this, T7 and Escherichia coli RNA polymerases transcribe the circles by a rolling mechanism, producing long concatemeric RNAs (approximately 7,500 nt). During the transcription reaction, the repeating RNAs self-cleave, ultimately reaching monomer length. Despite having self-complementary sequences at their substrate-binding domains, these monomeric 83-nt RNAs are shown to be catalytically active ribozymes that sequence-specifically cleave RNA targets in trans. In addition, a circular vector encoding a repeating (non-self-processing) ribozyme is described; the resulting multimeric ribozyme, targeted to a sequence in the HIV-1 genome, is also catalytically active in trans. This novel approach to the synthesis of catalytic RNAs offers a number of differences and potential advantages over current approaches to RNA synthesis.

DNA, Circular↗
PubMed1996

Non-hydrogen bonding 'terminator' nucleosides increase the 3'-end homogeneity of enzymatic RNA and DNA synthesis.

We report the use of novel non-polar nucleoside analogues as terminators of enzymatic RNA and DNA synthesis. Standard 'runoff' RNA synthesis by T7 RNA polymerase gives RNA products which have ragged ends as a result of transcription which often extends beyond the end of the template DNA strand. Similarly, the Klenow fragment of Escherichia coli DNA polymerase I tends to run past the end of the template strand during DNA synthesis. We report here that certain non-hydrogen-bonding nucleoside analogues, when placed at the downstream 5'-end of a template DNA strand, cause the polymerases to stop more abruptly at the last coding nucleotide. This results in a considerably more homogeneous oligonucleotide being produced. Three novel nucleosides are tested as potential terminators: 4-methylindole beta-deoxynucleoside (M), 1-naphthyl alpha-deoxynucleoside (N) and 1-pyrenyl alpha-deoxynucleoside (P). Comparison is made to an abasic nucleoside (phi) and to unterminated synthesis. Of these, M is found to be the most efficient at terminating transcription, and both P and M are highly effective at terminating DNA synthesis. It is also found that the ability of a nucleoside to stall synthesis when it is internally placed in the template strand is not necessarily a good predictor of terminating ability at the end of a template. Such terminator nucleosides may be useful in the preparative enzymatic synthesis of RNA and DNA, rendering purification simpler and lowering the cost of synthesis by preventing the uptake of potentially costly nucleotides into unwanted products.

Base Sequence↗
PubMed1996

Circular oligonucleotides: new concepts in oligonucleotide design.

Recent progress in the synthesis and properties of circular oligonucleotides as ligands for DNA and RNA and as templates for polymerase enzymes is described. Small synthetic circular DNAs, RNAs, and chimeric analogues ranging from 28 to 74 nucleotides in size have been synthesized with the use of nonenzymatic ligation strategy. Some of these were designed to undergo triplex formation with single-stranded DNA and RNA targets, and many bind with affinities and sequence selectivities considerably greater than those seen for linear oligonucleotides. Design strategies and modes of binding are discussed in the light of possible use of such molecules as hybridization probes, molecular diagnostics, and sequence-specific inhibitors of gene expression. Small circular oligonucleotides have also been shown to act as unusually efficient templates for DNA and RNA polymerases, which produce long, repeating copies of the circular sequence by a rolling circle process.

DNA↗
PubMed1995

Convergent DNA synthesis: a non-enzymatic dimerization approach to circular oligodeoxynucleotides.

We report a novel convergent approach to the construction of circular DNA oligonucleotides from two smaller linear precursors. Circular DNAs 34-74 nucleotides (nt) in size are constructed non-enzymatically in a single step from two half-length oligomers. A DNA template is used to assemble the constituent parts into a triple helical complex which brings the four reactive ends together for chemical ligation with BrCN/imidazole/Ni2+. A homodimerization reaction strategy is successfully used on a small scale to construct circles 42, 58 and 74 nt in size. In addition, a heterodimerization strategy is successfully used in two cases to construct circular 34mers from different 16mer and 18mer precursors. Measurement of preparative yields for one biologically active 34mer circle shows that the dimerization strategy gives a yield higher than that from conventional cyclization and nearly as high as that for a normally synthesized linear DNA, establishing that there is not necessarily a yield penalty for circle construction. Six additional preparative circle constructions, giving conversions of approximately 33-85% from precursors to circular product, are also described. Convergent strategies allow the construction of medium and large size DNA molecules in higher yields than can be achieved by standard linear synthesis alone.

Base Sequence↗
PubMed1995

Targeting pyrimidine single strands by triplex formation: structural optimization of binding.

Recent reports describe a new strategy for the binding of single-stranded pyrimidine sequences by triple helix formation. In this approach, a double-length purine-rich oligonucleotide binds a target strand, folding back to form an antiparallel pur.pur.pyr triple helix. We now describe a series of studies in which sequence and structural variations are made in such purine-rich ligands, in an effort to optimize binding properties. Comparison is made between the use of two separate strands and the use of single two-domain ligands; the latter are found to bind more tightly and to aggregate less in media containing Na+ or K+. Placement of mismatched bases in the target shows that sequence selectivity of binding is as high as that for Watson-Crick duplex formation. Variation of the lengths and sequences of loops bridging the binding domains demonstrates that dinucleotide loops composed of pyrimidines give the highest stability. Oligoethylene glycol-derived loop replacements are shown to give good binding affinity as well. The binding of an RNA target is shown to occur with the same affinity as the binding of DNA. In general, it is found that circular variants bind more tightly than do either separate strands or singly-linked ligands and unlike linear oligomers, the circular compounds do not aggregate to a large extent even in buffers containing 100 mM K+. Such structurally optimized ligands are useful in expanding the number of possible naturally-occurring sequences which can be targeted by triplex formation.

Base Sequence↗
PubMed1995

Origins of high sequence selectivity: a stopped-flow kinetics study of DNA/RNA hybridization by duplex- and triplex-forming oligonucleotides.

Stopped-flow UV kinetics and thermal denaturation experiments are used to examine the origins of high sequence selectivity and binding affinity of circular triplex-forming oligonucleotides with single-stranded DNA/RNA targets. These 34-nt probes are hybridized to a series of 12-nt target sequences which are fully complementary or which contain a single mismatch. Also studied for comparison are standard 12-nt Watson-Crick DNA or RNA complements. Several novel findings are described: (1) Circular triplex-forming oligomers bind targets with very high thermodynamic selectivity (up to 8-10 kcal/mol against a single-nucleotide mismatch), while linear strands show only 2-3 kcal/mol selectivity. (2) Rates for triplex formation by circular ligands are much greater than other reported triplex formation modes and are nearly the same as for Watson-Crick duplex formation. (3) DNA-DNA and RNA-RNA hybridization rates are similar for both duplex and triplex formation. (4) For both modes of binding, hybridization rates do not vary when a mismatch is introduced into the target, and, therefore, binding selectivity is reflected in large variations in dissociation, rather than association rates. Finally, (5) binding selectivity of circular ligands becomes significantly greater as pH is lowered; results indicate that the high sequence selectivity of the circular DNA ligand is due in large part to the special stability of the protonated C+G-C triad relative to unprotonated mismatched triads. The results are useful in the understanding of properties of nucleic acid complexes in general and give insight into optimum design for synthetic DNA-binding ligands.

Base Sequence↗