Biomedical subjects

L W McLaughlin

Publications and source records attributed to L W McLaughlin.

At least 55 records · Page 3Linked to original sources

PubMed1992

Fluorescence resonance energy transfer between specific-labeled sites on DNA.

Fluorescence resonance energy transfer on DNA has been studied for the estimation of distances between specific sites. Two kind of fluorophores, donor and acceptor, were incorporated on double-stranded DNA via phosphorothioate linkage (Sp, Rp, or racemic mixture). The thermal stability of labeled DNA's was slightly dependent on the stereochemical orientation of fluorophore, however all of the duplex structures were stable under the conditions for fluorescence study. The distances between donor and acceptor fluorophores, estimated from fluorescence energy transfer, generally agreed with the expected distance in a B-type DNA for the limiting distance.

Base Sequence↗

PubMed1991

Thermodynamic and structural properties of pentamer DNA.DNA, RNA.RNA, and DNA.RNA duplexes of identical sequence.

Four pentamers with the general sequence 5'CU(T)GU(T)G/5'CACAG have been prepared by chemical synthesis in order to generate duplex structures with common sequences. The four duplexes studied include the DNA.DNA duplex (5'dCACAG/5'dCTGTG) and the RNA.RNA duplex (5'rCUGUG/5'rCACAG) as well as the two corresponding DNA.RNA heteroduplexes (5'rCUGUG/5'dCACAG and 5'CACAG/5'dCTGTG). The measured entropy, enthalpy, and free energy changes upon melting are reported for each pentamer and compared to the predicted values where possible. Results show that the two DNA.RNA heteroduplexes are destabilized (delta G degrees 25 = -4.2 +/- 0.4 kcal/mol) relative to either the DNA.DNA duplex (delta G degrees 25 = -4.8 +/- 0.5 kcal/mol) or the RNA.RNA duplex (delta G degrees 25 = -5.8 +/- 0.6 kcal/mol). Circular dichroism spectra indicate that the RNA and the two heteroduplexes adopt an A-form conformation, while the DNA conformation is B-form. Imino proton NMR spectra also show that the heteroduplex structures resemble the RNA.RNA duplex.

Base Sequence↗

PubMed1991

The highly homologous isoschizomers RsrI endonuclease and EcoRI endonuclease do not recognize their target sequence identically.

Using a series of decadeoxyribonucleotides containing base analogues as substrates we measured the steady-state kinetic parameters for the reaction catalyzed by RsrI endonuclease and compared the results to those with its isoschizomer EcoRI. The kinetics of RsrI cleavage are affected by each substitution, with the effects being generally more deleterious than with EcoRI, as shown by the greater reduction in the specificity constant kcat/KM. The magnitudes of the effects of several substitutions are consistent with the formation of direct enzyme-nucleobase contacts at the indicated positions. With substrates containing 2-amino-purine or 2,6-diaminopurine at the central adenine or uracil at the outermost thymine in the recognition sequence, cleavage by RsrI was very slow, less than one-tenth the rate of the corresponding EcoRI-catalyzed reaction. The lower tolerance of RsrI endonuclease for functional group changes in its recognition site may reflect differences in the mechanisms of DNA recognition by the two enzymes. Although RsrI and EcoRI endonucleases bind with similar affinities to specific and nonspecific DNA sequences and appear to introduce similar structural distortions in DNA upon binding, the use of substrate analogues reveals significant differences at the level of catalysis in the mechanisms by which these two endonucleases recognize the duplex sequence GAATTC.

Base Sequence↗

PubMed1991

Properties of a U1/mRNA 5' splice site duplex containing pseudouridine as measured by thermodynamic and NMR methods.

Three RNA undecamers, 5'AUAC psi psi ACCUG (psi = pseudouridine), 5'AUACUUACCUG, and their complementary 11-mer 5'CAGGUAAGUAU, have been chemically synthesized by phosphite triester chemistry on a controlled-pore glass (CPG) support. The two duplexes formed with these molecules, 5'AUAC psi psi ACCUG/5'CAGGUAAGUAU and 5'AUACUUACCUG/5'CAGGUAAGUAU, represent the 5' end of human U1 snRNA paired to the mRNA consensus 5' splice site. In one undecamer, pseudouridines are incorporated at those positions corresponding to the native in vivo U1 snRNA, while the other (control) undecamer contains only uridine. Surprisingly, the NMR data show that the extra imino proton of the pseudouridines, which is found in the major groove and is presumably not hydrogen bonded, is clearly visible in the imino proton NMR spectrum at pH 6. This result suggests that the structure of the RNA restricts access of solvent to the major groove, slowing the exchange of the pseudouridine NH1 imino proton. A comparison of the thermodynamic properties of the two duplexes show that the free energy of duplex formation is unchanged by the substitution of pseudouridine for uridine.

Base Sequence↗

PubMed1991

Binding of Hoechst 33258 and 4',6'-diamidino-2-phenylindole to self-complementary decadeoxynucleotides with modified exocyclic base substituents.

Fluorescence titrations have been carried out to determine the association constants (Ka) for binding of the dyes Hoechst 33258 and DAPI to the self-complementary decamer d(CTGAATTCAG) and nine duplex derivatives with exocyclic substituent changes in the six central base pairs. Many Ka values are in the range (2-5) x 10(8) (duplex M)-1 at 5.5 degrees C. Replacement of the leftmost adenine by 2-aminopurine in the sequence decreases Ka for Hoechst 33258 by a factor of 170. When the centermost adenine is replaced by 2-aminopurine, Ka for Hoechst 33258 and DAPI is too small to be evaluated. When the centermost adenine is replaced by purine, Ka for both dyes increases, but this very stable duplex-Hoechst 33258 complex is nonfluorescent. The measured affinities are compared to expectations derived from X-ray studies with dodecamer-dye complexes having an identical central binding sequence (Pjura et al., 1987; Teng et al., 1988; Larsen et al., 1989).

2-Aminopurine↗

PubMed1990

Base stacking and unstacking as determined from a DNA decamer containing a fluorescent base.

Time-resolved fluorescence decay of a single-stranded DNA decamer d(CTGAAT5CAG), where d5 is the fluorescent base 1-(beta-D-2'-deoxyribosyl)-5-methyl-2-pyrimidinone, was measured and analyzed at several temperatures. The d5 base in the decamer is resolved into three states according to their fluorescence decay lifetime characteristics and temperature dependence of their associated amplitudes: fully extended and completely unstacked state, loosely associated state, and fully stacked state. These states are in slow exchange compared to their fluorescence decay rates. The population of the fully extended and completely unstacked state is small and decreases further with increasing temperature. The loosely associated state, whose fluorescence can still be efficiently quenched by other DNA bases, occupies a large portion of the conventionally defined unstacked state. Stacking enthalpy and entropy for the d5 base with thymine or cytosine bases in the DNA decamer are calculated to be -6.6 kcal/mol and -22 cal/mol.K, respectively. This work shows that fluorescent bases in DNA can be useful to the study of local conformations of bases.

Base Sequence↗

PubMed1990

A demonstration of the intrinsic importance of stabilizing hydrophobic binding and non-covalent 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)

Animals↗

PubMed1989

Structure and dynamics of a fluorescent DNA oligomer containing the EcoRI recognition sequence: fluorescence, molecular dynamics, and NMR studies.

The self-complementary DNA decamer duplex d(CTGAATTCAG)2 and its modified counterpart d(CTGA[2AP]TTCAG)2, where the innermost adenine (6-aminopurine) has been replaced with the fluorescent analogue 2-aminopurine (2AP), have been studied by fluorescence and NMR spectroscopy and simulated by molecular dynamics. Both decamers are recognized and cleaved by the EcoRI restriction endonuclease. 2D NMR results show that both decamers have a standard B-type conformation below 20 degrees C, though a disturbance exists to the 5' side of the 2AP site which may originate from increased local mobility. The fluorescence and fluorescence anisotropy decays of both decamers, as well as the one containing 2AP in only one chain, were studied as a function of temperature. The data show that the 2AP base exists in a temperature-dependent distribution of states and shows rapid motions, suggesting interconversion among these states on a time scale of about 10(-10) s. The integrated fluorescence of the decamer with 2AP in both chains shows a large increase around the helix melting temperature whereas the decamer with one 2AP shows only a mild increase, showing that the mixed helix has a different structural transition as sensed by the 2AP base. The data suggest a model of conformational states which have distinct fluorescence decay times. The various states may differ in the degree of base stacking. Fluctuations in the degree of stacking of the A or 2AP base are supported by molecular dynamics simulations, which additionally show that the 2AP-T or A-T base pair hydrogen bonds remain intact during these large motions.

DNA↗

PubMed1989

Effects of functional group changes in the EcoRV recognition site on the cleavage reaction catalyzed by the endonuclease.

Oligodeoxynucleotides have been prepared which contain changes in the functional group pattern present in the EcoRV recognition site d(GATATC). These modifications involve the deletion of specific functional groups or the reversal of the relative positions of functional groups within the canonical six base pair recognition site. The duplex stability of these modified oligodeoxynucleotides has been assessed by determining the thermodynamic parameters characterizing helix formation. Steady-state kinetic parameters have been used to characterize the interaction of the modified oligodeoxynucleotides with the EcoRV endonuclease. The enzyme is very sensitive to the deletion of either of the adenine amino or thymine methyl groups, or the reversal of the relative positions of the adenine amino group and thymine carboxy group which form an interstrand hydrogen bond in the major groove of the B-DNA helix. Conversely, deletion of the guanine amino group had only minimal effects upon the measured kinetic parameters. Deletion of the exocyclic amino group from the "inner" dA-dT base pair resulted in the fragment which interacted with the enzyme on the basis of observed inhibition experiments but was not cleaved. The results suggest that the endonuclease interacts with its recognition sequence via contacts in the major groove of the B-DNA helix and that both hydrogen bonding to the adenine amino groups and also hydrophobic interactions with the thymine methyl groups are involved.

Adenine↗

PubMed1989

The synthesis of 2-pyrimidinone nucleosides and their incorporation into oligodeoxynucleotides.

The synthesis of 1-(beta-D-2'-deoxyribosyl)-2-pyrimidinone (dK) and its 5-methyl derivative (d5) from 2'-deoxycytidine or 2'-deoxythymidine, respectively, via silver oxide oxidation of 4-hydrazinopyrimidines is described. The necessary hydrazine substituted pyrimidine nucleosides have been prepared by transamination of a protected cytidine derivative or by addition/elimination reactions to an O4-sulfonated thymidine derivative. Oxidation of the 4-hydrazino pyrimidines was complicated by a competing hydrolytic reaction which generated 2'-deoxyuridine or 2'-deoxythymidine. However, in the presence of an organic base such as triethylamine, oxidation became the predominant reaction. After suitable protection and formation of the 3'-phosphoramidite derivatives, these modified nucleosides were incorporated into seven self-complementary oligodeoxynucleotides by chemical synthesis using phosphite triester methodology. Oligodeoxynucleotides were prepared such that dA-dT and dG-dC base pairs were substituted by dA-d5 or dG-dK base pairs, respectively. Both circular dichroism spectra and thermal denaturation studies were used to characterize the modified oligodeoxynucleotides.

Base Sequence↗

PubMed1989

"Post-assay" covalent labeling of phosphorothioate-containing nucleic acids with multiple fluorescent markers.

A simple protocol has been developed which allows the covalent introduction of multiple fluorescent markers into DNA fragments after gel electrophoresis techniques, that is, while the nucleic acid is imbedded in the polyacrylamide gel matrix. "Post-assay" fluorescent labeling in this manner employs DNA fragments containing phosphorothioate diesters, which can be easily incorporated during chemical and enzymatic synthesis procedures, and can be alkylated with the fluorescent marker monobromobimane. Labeling the internucleotidic phosphorus residue in this manner allows the introduction of a fluorescent marker for each nucleotide residue present. Roughly a linear increase in emitted fluorescence, thus detection sensitivity, is observed with an increasing number of bimane markers. With this technique, oligodeoxynucleotides and DNA fragments can be observed in the gel matrix, without sophisticated electronic detection devices in the low femtomolar (10(-15) mol) range.

Bridged Bicyclo Compounds↗

PubMed1989

The introduction of reporter groups at multiple and/or specific sites in DNA containing phosphorothioate diesters.

DNA fragments containing phosphorothioate diesters provide nucleophilic sites which are amenable to labeling by spin labels or fluorophores. Selecting the position for an individual phosphorothioate diester allows highly specific placement of the reporter group. The substitution of a phosphorothioate diester for each and every internucleotidic phosphodiester allows the incorporation of multiple reporter groups; ideally one for each nucleoside residue. With the use of multiple fluorophores a post-assay fluorescent labeling technique has been developed which allows the detection of DNA fragments with the "naked-eye" in the low femtomolar (10(-15) moles) range.

Bridged Bicyclo Compounds↗

PubMed1988

An oligodeoxynucleotide affinity column for the isolation of sequence specific DNA binding proteins.

A nucleic acid affinity matrix containing a short oligodeoxynucleotide ligand has been prepared as an example of a material which can be used for the rapid and effective isolation of sequence specific DNA binding proteins. Two complementary oligodeoxynucleotides have been employed, one of which contains a small 5'-spacer arm with a terminal thiol group. Using this terminal thiol group, the ligand can be covalently coupled to Tresyl-activated Sepharose 4B or Epoxy-activated Sepharose 6B via a thioether linkage. This approach allows the specific attachment of the nucleic acid ligand via its 5'-terminus to the insoluble matrix. The double stranded affinity material was obtained by annealing of the complementary DNA fragment. As an example, we have used an eicosomer affinity column containing the sequence d(GAATTC) for the isolation of the Eco RI restriction endonuclease. Using a single column, the enzyme could be isolated by eluting the column with a single step or multistep gradient of increasing salt concentration. The enzyme was purified to 75%-85% homogeneity with yields of 0.1 mg to 0.2 mg from 0.5 g of cell paste.

Base Sequence↗

PubMed1988

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.

Base Sequence↗

PubMed1988

A new approach to the synthesis of a protected 2-aminopurine derivative and its incorporation into oligodeoxynucleotides containing the Eco RI and Bam HI recognition sites.

A protected 2-aminopurine nucleoside suitable for incorporation into oligodeoxynucleotides using phosphite triester chemical synthesis procedures has been prepared via oxidation of a purine hydrazino derivative with silver (I) oxide. Five oligodeoxynucleotides containing Eco RI and Bam HI recognition sites have been prepared such that, in the double stranded form, the 2-aminopurine base has either a complementary thymine or cytosine nucleobase. The helix character and thermodynamic parameters for helix formation have been examined.

2-Aminopurine↗

PubMed1988

Refinement of the solution structure of the DNA dodecamer 5'd(CGCGPATTCGCG)2 containing a stable purine-thymine base pair: combined use of nuclear magnetic resonance and restrained molecular dynamics.

The solution structure of the self-complementary dodecamer 5'd(CGCGPATTCGCG)2, containing a purine-thymine base pair within the hexameric canonical recognition site GAATTC for the restriction endonuclease EcoRI, is investigated by nuclear magnetic resonance spectroscopy and restrained molecular dynamics. Nonexchangeable and exchangeable protons are assigned in a sequential manner. A set of 228 approximate interproton distance restraints are derived from two-dimensional nuclear Overhauser enhancement spectra recorded at short mixing times. These distances are used as the basis for refinement using restrained molecular dynamics in which the interproton distance restraints are incorporated into the total energy function of the system in the form of effective potentials. Eight calculations are carried out, four starting from classical A-DNA and four from classical B-DNA. In all cases convergence to very similar B-type structures is achieved with an average atomic root mean square (rms) difference between the eight converged structures of 0.7 +/- 0.2 A, compared to a value of 6.5 A for that between the two starting structures. It is shown that the introduction of the purine-thymine mismatch does not result in any significant distortion of the structure. The variations in the helical parameters display a clear sequence dependence. The variation in helix twist and propeller twist follows Calladine's rules and can be attributed to the relief of interstrand purine-purine clash at adjacent base pairs. Overall the structure is straight. Closer examination, however, reveals that the central 5 base pair steps describe a smooth bend directed toward the major groove with a radius of curvature of approximately 38 A, which is compensated by two smaller kinks in the direction of the minor groove at base pair steps 3 and 9. These features can be explained in terms of the observed variation in roll and slide.

Base Composition↗

PubMed1987

Effects of functional group changes in the EcoRI recognition site on the cleavage reaction catalyzed by the endonuclease.

Oligodeoxynucleotides have been prepared that contain changes in the functional group pattern present in the EcoRI recognition site. These changes involve "functional group deletions", "functional group reversals", and "displaced functional groups". Steady-state kinetic parameters have been used to characterize the interaction of these modified recognition sites with the EcoRI endonuclease. Changes in the functional group pattern have varying effects upon the cleavage reaction. Both the exocyclic amino groups of the two adenine residues and the methyl groups of the thymine residues appear to interact with the endonuclease quite differently. In both cases efficient catalysis was observed when these functional groups were present at the "outer" dA-dT base pair. Selectivity was decreased by over an order of magnitude largely via increases in Km when these functional groups were deleted. Similar modifications at the "inner" dA-dT base pair did not alter the kinetic parameters significantly from those observed with the native sequence. Addition of an amino group to the minor groove at the outer dA-dT base pair resulted in a modified recognition site that interacted with the enzyme, on the basis of observed competitive inhibition kinetics, but was not cleaved.

Base Sequence↗

PubMed1987

Base pair opening dynamics of a 2-aminopurine substituted Eco RI restriction sequence and its unsubstituted counterpart in oligonucleotides.

Studies of 1H NMR selective saturation recovery were performed to determine the imino proton exchange with solvent water of the base pairs in the Eco RI endonuclease recognition sequence GAATTC, placed at the center of self-complementary decamer and dodecamer oligonucleotides. In one oligonucleotide the innermost adenine was replaced by the fluorescent base analogue 2-aminopurine (2AP). From the measurements at different concentrations of TRIS buffer acting as proton exchange catalyst, base pair lifetimes were evaluated. The results at 25 degrees show that the AT base pairs have lifetimes of the order of a few ms, whereas the surrounding GC base pairs in a dodecamer have lifetimes of about 100 ms. The (2AP)T base pair has a shorter lifetime than the corresponding AT base pair. The temperature dependent optical absorption, and for the 2AP containing oligonucleotide fluorescence, were used to study the single strand-duplex equilibrium of the decamers. The results indicate that NMR and the optical techniques, although applied at very different concentrations, monitor the same conformational transition of the oligonucleotide.

2-Aminopurine↗