Thermodynamic, spectroscopic, and equilibrium binding studies of DNA sequence context effects in six 22-base pair deoxyoligonucleotides.

Effects of different end sequences on stability, circular dichroism spectra (CD), and enzyme binding properties were investigated for six 22-base pair, non-self-complementary duplex DNA oligomers. The center sequences of these deoxyoligonucleotides have 8-14 base pairs in common and are flanked on both sides by sequences differing in context and A-T content. Temperature-induced melting transitions monitored by differential scanning calorimetry (DSC) and ultraviolet absorbance were measured for the six duplexes in buffered 115 mM Na(+) solutions. Values of the melting transition enthalpy, DeltaH(cal), and entropy, DeltaS(cal), were obtained directly from DSC experiments. Melting transition parameters, DeltaH(vH) and DeltaS(vH), were also estimated from van't Hoff analysis of optical melting curves collected as a function of DNA concentration, assuming a two-state melting transition. Melting free energies (20 degrees C) of the six DNAs evaluated from DSC experiments ranged from -18.7 to -32.7 kcal/mol. van't Hoff estimates of the free energies ranged from -18.5 to -48.0 kcal/mol. With either method, the trends in free energy as a function of sequence were identical. Equilibrium binding by BamHI restriction endonuclease to the 22-base pair DNAs was also investigated. The central eight base pairs of all six molecules, 5'-A-GGATCC-A-3', contained a BamHI recognition sequence bounded by A-T base pairs. Magnesium free binding assays were performed by titering BamHI against a constant concentration of each of the deoxyoligonucleotide substrates and analyzing reaction products by gel retardation. Binding isotherms of the total amount of bound DNA versus protein concentration were constructed which provided semiquantitative estimates of the equilibrium dissociation constants for dissociation of BamHI from the six DNA oligomers. Dissociation constants ranged from 0.5 x 10(-)(9) to 12.0 x 10(-)(9) M with corresponding binding free energies of -12.5 to -10.6 (+/-0. 1) kcal/mol. An inverse relationship is found when binding and stability are compared.

Melting studies of short DNA hairpins: influence of loop sequence and adjoining base pair identity on hairpin thermodynamic stability.

Spectroscopic and calorimetric melting studies of 28 DNA hairpins were performed. These hairpins form by intramolecular folding of 16 base self-complementary DNA oligomer sequences. Sequence design dictated that the hairpin structures have a six base pair duplex linked by a four base loop and that the first five base pairs in the stem are the same in every molecule. Only loop sequence and identity of the duplex base pair closing the loop vary for the set of hairpins. For these DNA samples, melting studies were carried out to investigate effects of the variables on hairpin stability. Stability of the 28 oligomers was ascertained from their temperature-induced melting transitions in buffered 115 mM Na(+) solvent, monitored by ultraviolet absorbance and differential scanning calorimetry (DSC). Experiments revealed the melting temperatures of these molecules range from 32.4 to 60.5 degrees C and are concentration independent over strand concentrations of 0.5 to 260 microM; thus, as expected for hairpins, the melting transitions are apparently unimolecular. Model independent thermodynamic transition parameters, DeltaH(cal), DeltaS(cal), and DeltaG(cal), were determined from DSC measurements. Model dependent transition parameters, DeltaH(vH), DeltaS(vH), and DeltaG(vH) were estimated from a van't Hoff (two-state) analysis of optical melting transitions. Results of these studies reveal a significant sequence dependence to DNA hairpin stability. Thermodynamic parameters evaluated by either procedure reveal the transition enthalpy, DeltaH(cal) (DeltaH(vH)) can differ by as much as 20 kcal/mol depending on sequence. Similarly, values of the transition entropy DeltaS(cal) (DeltaS(vH)) can differ by as much as 60 cal/Kmol (eu) for different molecules. Differences in free energies DeltaG(cal) (DeltaG(vH)) are as large as 4 kcal/mol for hairpins with different sequences. Comparisons between the model independent calorimetric values and the thermodynamic parameters evaluated assuming a two-state model reveal that 10 of the 28 hairpins display non-two-state melting behavior. The database of sequence-dependent melting free energies obtained for the hairpins was employed to extract a set of n-n (nearest-neighbor) sequence dependent loop parameters that were able to reproduce the input data within error (with only two exceptions). Surprisingly, this suggests that the thermodynamic stability of the DNA hairpins can in large part be reasonably represented in terms of sums of appropriate nearest-neighbor loop sequence parameters.

The thermodynamic advantage of DNA oligonucleotide 'stacking hybridization' reactions: energetics of a DNA nick.

'Stacking hybridization reactions' wherein two or more short DNA oligomers hybridize in a contiguous tandem orientation onto a longer complementary DNA single strand have been employed to enhance a variety of analytical oligonucleotide hybridization schemes. If the short oligomers anneal in perfect head-to-tail register the resulting duplex contains a nick at every boundary between hybridized oligomers. Alternatively, if the short oligomers do not hybridize precisely in register, i.e. single strand regions on the longer strand are left unbound, gaps are formed between regions where short oligomers bind. The resulting gapped DNA duplexes are considerably less stable than their nicked duplex analogs. Formation of base pair stacking interactions between neighboring oligomers at the nicks that do not occur in gapped duplexes has been proposed as the source of the observed added stability. However, quantitative evidence supporting this hypothesis for DNA has not been reported. Until now, a direct comparison of the thermodynamics of DNA nicks versus DNA gaps has not been performed. In this communication we report such a comparison. Analysis of optical melting experiments in a well defined molecular context enabled quantitative evaluations of the relative thermodynamic difference between nicked and gapped DNA duplexes. Results of the analysis reveal that a nick may be energetically favored over a gap by at least 1.4 kcal/mol and perhaps as much as 2.4 kcal/mol. The presence of a 5'phosphate at a nick or gap fails to significantly affect their stabilities.

Regional localization of the putative cell surface receptor for HTLV-I to human chromosome 17q23.2-17q25.3.

The gene for the cell surface receptor for HTLV-I, the etiologic agent of adult T-cell leukemia and HTLV-I-associated myelopathy, has been localized to distal human chromosome 17q. A panel of somatic cell hybrids containing fragments of human 17q as the only human genetic component was mapped with a set of 10 chromosome 17 probes and utilized to regionally localize the gene. When compared to the murine fibroblast fusion partner, L-M(TK-), and a hybrid cell line containing human chromosome 20, human 17q-containing hybrid cells bound high levels of both HTLV-I virions and the monoclonal antibody, Mab 34-23, which may be directed against the putative HTLV-I receptor. Additional experiments revealed that the human 17q-containing hybrids could also be more efficiently infected by cell-free HTLV-I virions than could the control cell lines. Western blot analyses of cell lysates showed that recombinant HTLV-I envelope gp46 protein and Mab 34-23 both bound to proteins of approximate MW 30 and 31 kDa which were found only in the hybrid cell lines which contained human chromosome 17q. The data suggest that the gene for the HTLV-I receptor is located on the distal region of human chromosome 17q demarcated by the tk-1 locus (17q23.2-17q25.3).

Variation in genomic Alu repeat density as a basis for rapid construction of low resolution physical maps of human chromosomes.

Human DNA restriction fragments containing high numbers of Alu repeat sequences can be preferentially detected in the presence of other human DNA restriction fragments in DNA from human: rodent somatic cell hybrids when the DNA is fragmented with enzymes that cleave mammalian DNA infrequently. This ability to lower the observed human DNA complexity allowed us to develop an approach to order rapidly somatic hybrid cell lines retaining overlapping human genomic domains. The ordering process also generates a relative physical map of the human fragments detected with Alu probe DNA. This process can generate physical mapping information for human genomic domains as large as an entire chromosome (100,000 kb). The strategy is demonstrated by ordering Alu-detected NotI fragments in a panel of mouse: human hybrid cells that span the entire long arm of human chromosome 17.

An efficient technique for obtaining sequences flanking inserted retroviruses.

Genomic mapping studies frequently employ retrovirus-mediated transfer of dominant selectable markers to specific target chromosomes. DNA probes containing sequences adjacent to inserted proviruses are valuable mapping tools in such studies. We have implemented a strategy for amplification of chromosomal sequences flanking the 5' LTR of MoMuLV-based vectors. Probes derived from these amplification products successfully differentiated murine versus human proviral localization in retrovirus-infected mouse-human chromosome 17q hybrid cells.