Larry W. McLaughlin

Nucleic acid resolution by mixed-mode chromatography

Two approaches to the preparation of mixed-mode (ionic—hydrophobic) supports for high-performance liquid chromatography are described. In the first, hydrophobic moieties are covalently bound to an anion-exchange support (ASP-Hypersil). In the second approach ionic amines are bound through hydrophobic interactions to a reversed-phase support (ODS-Hypersil). In the former case the hydrophobicity of the support is controlled by addition of alkyl groups of various chain lengths and ionic interactions are controlled by the pH of the mobile phase. The analysis of two simple solutes, (Ap)3A and (Up)3U, shows that both ionic and hydrophobic interactions are present. In the latter case the support is shown to be useful for the resolution of large oligonucleotides such as (Up)89 from (Up)90. Hydrophobic interactions are important in the observed separations. The binding of a non-polar amino acid to a particular tRNA results in a large shift in retention volume as a result of a stronger adsorption on the support.

An in Vitro Selection System for TNA

(3‘-2‘)-α-l-Threose nucleic acid (TNA) is an unnatural polymer that possesses the rare ability to base-pair with RNA, DNA, and itself. This feature, coupled with its chemical simplicity, makes TNA of interest as a possible progenitor of RNA during the early history of life. To evaluate the functional potential of TNA, we have developed a system for the in vitro selection of TNA. We identified the Therminator DNA polymerase as a remarkably efficient DNA-dependent TNA polymerase capable of polymerizing more than 50 tNTPs. We have also developed a method of covalently linking a DNA template to the TNA strand that it encodes, thus obviating the need for a TNA-dependent DNA polymerase during cycles of selection.

High fidelity TNA synthesis by Therminator polymerase

Therminator DNA polymerase is an efficient DNA-dependent TNA polymerase capable of polymerizing TNA oligomers of at least 80 nt in length. In order for Therminator to be useful for the in vitro selection of functional TNA sequences, its TNA synthesis fidelity must be high enough to preserve successful sequences. We used sequencing to examine the fidelity of Therminator-catalyzed TNA synthesis at different temperatures, incubation times, tNTP ratios and primer/template combinations. TNA synthesis by Therminator exhibits high fidelity under optimal conditions; the observed fidelity is sufficient to allow in vitro selection with TNA libraries of at least 200 nt in length.

Kinetic Analysis of an Efficient DNA-Dependent TNA Polymerase

α-l-Threofuranosyl nucleoside triphosphates (tNTPs) are tetrafuranose nucleoside derivatives and potential progenitors of present-day β-d-2‘-deoxyribofuranosyl nucleoside triphosphates (dNTPs). Therminator DNA polymerase, a variant of the 9°N DNA polymerase, is an efficient DNA-directed threosyl nucleic acid (TNA) polymerase. Here we report a detailed kinetic comparison of Therminator-catalyzed TNA and DNA syntheses. We examined the rate of single-nucleotide incorporation for all four tNTPs and dNTPs from a DNA primer−template complex and carried out parallel experiments with a chimeric DNA−TNA primer−DNA template containing five TNA residues at the primer 3‘-terminus. Remarkably, no drop in the rate of TNA incorporation was observed in comparing the DNA−TNA primer to the all-DNA primer, suggesting that few primer-enzyme contacts are lost with a TNA primer. Moreover, comparison of the catalytic efficiency of TNA synthesis relative to DNA synthesis at the downstream positions reveals a difference of no greater than 5-fold in favor of the natural DNA substrate. This disparity becomes negligible when the TNA synthesis reaction mixture is supplemented with 1.25 mM MnCl2. These results indicate that Therminator DNA polymerase can recognize both a TNA primer and tNTP substrates and is an effective catalyst of TNA polymerization despite changes in the geometry of the reactants.

Chemically synthesized hydrophobic anion-exchange high-performance liquid chromatography supports used for oligonucleotide resolution by mixed mode chromatography

A commercially available aminopropylsilyl bonded-phase high-performance liquid chromatography support has been modified with three different organic acids each containing an amine group and a hydrophobic moiety to produce anion-exchange supports with increased hydrophobicity. The chromatographic characteristics of these supports were examined using two oligonucleotides of equal chain length but differing hydrophobic character, (Ap)3A and (Up)3U. The resolution of these two solutes on the modified supports in the pH range 4.5–6.5 and an acetonitrile concentration of 0-48% is described. The most effective support contains a phenylalanine moiety (APS-PHE) bound through an amide to the commercial support. This support has been used to resolve oligonucleotides of the same size (6–12 residues) but different sequence prepared enzymatically or chemically. The difficulties inherent in parameter optimization in mixed-mode chromatography are discussed.

A demonstration of the intrinsic importance of stabilizing hydrophobic binding and non-convalent van der waals contacts dominant in the non-covalent CC-1065/B-DNA binding

The comparative DNA binding properties and cytotoxic activity of CDPIn methyl esters (n = 1-5) vs. PDE-In methyl esters (n = 1-3) are detailed in studies which provide experimental evidence for the intrinsic importance of stabilizing hydrophobic binding and non-covalent van der Waals contacts dominant in the CC-1065/B-DNA minor groove binding. High affinity minor groove binding to DNA was established through: (1) the observation of CDPI3 binding (UV) but not unwinding of supercoiled DNA (phi 174 RFI DNA) thus excluding intercalative binding; (2) the observation of CDPI3 binding to T4 phage DNA (UV, delta Tm) in which the major groove is occluded by glycosylation thus excluding major groove binding; (3) the observation of salt (Na+) concentration independent high affinity CDPI3 binding to poly(dA . poly(dT) thus excluding simple electrostatic binding to the DNA phosphate backbone; and further inferred through (4) the observation of an intense induced dichroism (ICD, poly(dA) . poly(dT) and poly(dG) . poly(dC) [phi]23(358) = 24,000 and 23,500). This high affinity minor groove binding is sufficient to produce a potent cytotoxic effect.(ABSTRACT TRUNCATED AT 400 WORDS)

tRNA separation by high-performance liquid chromatography using an aggregate of ODS-Hypersil and trioctylmethylammonium chloride.

High-performance liquid chromatography on a reversed-phase support treated with a tetraalkylammonium salt was used to separate tRNAs from bakers yeast. While resolution by this column appears to result from both anion-exchange and reversed-phase chromatography, it is the hydrophobic interactions which govern the separation of one tRNA from another. Chromatography of bulk tRNA resulted in a number of fractions with different amino acid acceptor activities and little cross-contamination. In some cases the column resolved several single nucleotide modifications of tRNAPhe. Using a 250 x 6.2 mm column it has been possible to chromatograph a minimum of 100 A260 units of tRNA without serious loss in resolution. tRNAs isolated from this column as the last step of a purification procedure have very high amino acid acceptor activities.

Isolation of specific tRNAs using an ionic-hydrophobic mixed-mode chromatographic matrix

The coating of a C18-reversed-phase high performance liquid chromatography support (octadecylsilyl-Hypersil) with a tetraalkylammonium salt (methyltrioctylammonium chloride) produces a chromatographic matrix with both ionic and hydrophobic character. Using this material oligonucleotides and tRNAs can be separated with high resolution. The observed resolution is in part due to the apparent lack of diffusion processes occurring during chromatography with this matrix. Some tRNAs can be obtained in high purity from a bulk tRNA mixture after a single chromatographic step. In general it is more efficient to use the matrix as the last step of a purification procedure for a particular tRNA. A two-step procedure is described which allows, in some cases, the isolation of small quantities of specific tRNA isoacceptors.

DNA curvature in native and modified EcoRI recognition sites and possible influence upon the endonuclease cleavage reaction.

The ligation of a decadeoxynucleotide containing the EcoRI recognition site forms a series of multimers which appear to be curved based on observed anomalous gel migration in polyacrylamide gels. The degree of DNA curvature present in the recognition sequence, based upon the observed migration anomaly, can be altered by modifications to the purine functional groups at the 2- and 6-positions. Deletion of the guanine 2-amino group, occurring in the minor groove of the B-DNA helix, is most effective in increasing the observed DNA curvature. Conversely, the displacement of an amino group from the major groove to the minor groove eliminates curvature. DNA curvature is also modulated by the exocyclic group at the purine 6-position with decreasing curvature observed when changing the amino group to a carbonyl or proton substituent. Differences in the kinetic parameters characterizing the cleavage reaction by the endonuclease for many of the modified sequences are the result of modifications of functional groups in the major groove, which are likely to contact the endonuclease during catalysis. However, with two examples, significant decreases in the observed specificity constant (kcat/Km), characterizing the protein-nucleic acid interaction, cannot be easily explained in terms of such functional group contacts. It is more likely in these cases that the functional group modifications affect the efficiency of the endonuclease-DNA interaction by modulation of the structure of the double-stranded DNA helix. With both examples, modifications have been made to minor groove substituents. The extent of DNA curvature is increased significantly for one and decreased for the other, compared with that observed for the native recognition site. The results suggest that curvature of the DNA helix axis is an intrinsic property of the d(GAATTC) sequence which helps to optimize the protein-nucleic acid interactions observed for the EcoRI restriction endonuclease.

Mild acid hydrolysis of 2-pyrimidinone-containing DNA fragments generates apurinic/apyrimidinic sites

Abstract Facile glycosidic bond cleavage of 2-pyrimidinone-2′-deoxynucleosides occurs under mildly acidic conditions (pH 3.0) and ambient temperature. When a 2-pyrimidinone nucleoside residue is present in a DNA fragment, hydrolysis results in an apurinic/apyrimidinic (abasic) site. The incorporation of 2-pyrimidinones into chemically synthesized DNA provides a route for the chemical generation of an abasic site at a preselected position in the sequence.