The design of an agent to bend DNA.

An artificial DNA bending agent has been designed to assess helix flexibility over regions as small as a protein binding site. Bending was obtained by linking a pair of 15-base-long triple helix forming oligonucleotides (TFOs) by an adjustable polymeric linker. By design, DNA bending was introduced into the double helix within a 10-bp spacer region positioned between the two sites of 15-base triple helix formation. The existence of this bend has been confirmed by circular permutation and phase-sensitive electrophoresis, and the directionality of the bend has been determined as a compression of the minor helix groove. The magnitude of the resulting duplex bend was found to be dependent on the length of the polymeric linker in a fashion consistent with a simple geometric model. Data suggested that a 50-70 degrees bend was achieved by binding of the TFO chimera with the shortest linker span (18 rotatable bonds). Equilibrium analysis showed that, relative to a chimera which did not bend the duplex, the stability of the triple helix possessing a 50-70 degrees bend was reduced by less than 1 kcal/mol of that of the unbent complex. Based upon this similarity, it is proposed that duplex DNA may be much more flexible with respect to minor groove compression than previously assumed. It is shown that this unusual flexibility is consistent with recent quantitation of protein-induced minor groove bending.

NOMAD: a versatile strategy for in vitro DNA manipulation applied to promoter analysis and vector design.

Molecular analysis of complex modular structures, such as promoter regions or multi-domain proteins, often requires the creation of families of experimental DNA constructs having altered composition, order, or spacing of individual modules. Generally, creation of every individual construct of such a family uses a specific combination of restriction sites. However, convenient sites are not always available and the alternatives, such as chemical resynthesis of the experimental constructs or engineering of different restriction sites onto the ends of DNA fragments, are costly and time consuming. A general cloning strategy (nucleic acid ordered assembly with directionality, NOMAD; WWW resource locator http:@Lmb1.bios.uic.edu/NOMAD/NOMAD.htm l) is proposed that overcomes these limitations. Use of NOMAD ensures that the production of experimental constructs is no longer the rate-limiting step in applications that require combinatorial rearrangement of DNA fragments. NOMAD manipulates DNA fragments in the form of "modules" having a standardized cohesive end structure. Specially designed "assembly vectors" allow for sequential and directional insertion of any number of modules in an arbitrary predetermined order, using the ability of type IIS restriction enzymes to cut DNA outside of their recognition sequences. Studies of regulatory regions in DNA, such as promoters, replication origins, and RNA processing signals, construction of chimeric proteins, and creation of new cloning vehicles, are among the applications that will benefit from using NOMAD.

Design of artificial sequence-specific DNA bending ligands.

Proteins that bend DNA are important regulators of biological processes. Sequence-specific DNA bending ligands have been designed that bind two noncontiguous sites in the major groove and induce a bend in the DNA. An oligonucleotide containing pyrimidine segments separated by a central variable linker domain simultaneously binds by triple helix formation two 15-bp purine tracts separated by 10 bp. Bend angles of 61 degrees, 50 degrees, and 38 degrees directed towards the minor groove were quantitated by phasing analysis for linkers of four, five, and six T residues, respectively. The design and synthesis of nonnatural architectural factors may provide a new class of reagents for use in biology and human medicine.