Edmond J. Gabbay

INTERACTION SPECIFICITY OF NUCLEIC ACIDS

Nuclear magnetic resonance provides a unique method for studying the interaction of small molecules with macromolecules. The influence of a macromolecule on the line width and the chemical shifts of the protons of a small molecule provide information concerning the degree to which motion of specific parts of the small molecule becomes restricted and concerning the magnetic environment of the small molecule when it is bound. For example, it is well known that if the rate of tumbling of molecules in solutions is lower than the typical Larmor frequencies Wo (of the order of 1OR-109 rad sec-l for protons in the conventional magnetic field), then T,, the transverse relaxation time, is considerably diminished, leading to substantial line broadening of the proton signa1.l This situation obtains if thc proton is contained in a rigid macromolecule, e.g. or if the proton is contained in a slowly tumbling small molecule bound to a macromolecule.^ Over the past year we have examined the interaction specificity of more than 100 polyammonium salts with nucleic acids by temperature-dependent nuclear-magnetic-resonance techniques. Threc basic types of interactions have been observed: ( 1) external electrostatic binding, ( 2 ) intercalation, and ( 3 ) strong external electrostatic and hydrophobic-type binding. These modes of interactions may be illustrated by reporter molecules 1-10, the first nine of which are demonstrated in FIGURE 1 .

Chapter 18 Intercalating Agents as Probes of Chromatin Structure

Publisher Summary This chapter focuses on intercalating agents as probes of chromatin structure. The intercalation model, proposed in the early 1960s, involves the unwinding of the deoxyribose sugar phosphate chains and elongation of the DNA molecule to produce an approximately 3.4 A-thick hydrophobic space to accommodate the insertion of planar aromatic molecules between base pairs. Lerman provided extensive experimental support for the intercalation model. A number of planar aromatic molecules have been shown to form intercalated complexes with DNA; these include the acridine dyes, antiparasitic drugs, antitumor agents, and a variety of small molecules. The structure of some typical intercalating molecules is shown in the chapter. A brief description of the characteristic physical and chemical changes occurring in both DNA and the bound small molecule as a result of intercalation are presented as an introduction to the more complex phenomena which occur with chromatin. Effects of intercalation on the physical properties of DNA are discussed. Proton magnetic resonance techniques are also described in the chapter.

Topography of nucleic acid helices in solutions. XXIV intercalation specificities of DNA and their possible role in the recognition process

Abstract Through the use of reporter molecules, I, it is shown that more than one type of intercalating site must exist in DNA. Although this finding is completely consistent with the DNA structure it has not yet been demonstrated. The significance of the presence of different intercalating sites in DNA may be of extreme importance in the recognition of nucleic acid-protein systems.

Topography of nucleic acid helices in solutions. XXVIII. Evidence for a dynamic structure of DNA in solution

Abstract Proton magnetic resonance (pmr), ultraviolet absorption, induced circular dichroism (CD), and viscometric evidence is presented which show that reporter molecules 1 and 2 bind to DNA via an intercalation process. Preliminary kinetic studies show that the DNA· 1 complex forms rapidly (i.e., 2 complex forms at a considerably slower rate ( t 1 2 > 100 msec ). The kinetic results, and the steric requirements for intercalation of 2 can be explained on the basis of a dynamic structure of DNA.

Preparation and characterization of an α-amanitin azo derivative with a free carboxyl group and its bovine serum albumin conjugate☆

Abstract α-Amanitin has been diazotized to p -aminobenzoylglycylglycine to form the stable derivative amanitinylazobenzoyl- N -glycylglycine (ABGG) with a free carboxyl group. The ABGG displayed a K I of 6.1 × 10 −9 m to calf thymus RNA polymerase II compared to a K I of 1.8 × 10 −9 m for α-amanitin. The ABGG was coupled to bovine serum albumin (BSA) with watersoluble carbodiimide to provide a covalent conjugate with 2.9 mol of ABGG per mole of BSA. The conjugation resulted in approximately 80% intermolecular crosslinking of protein, a 66% decrease in the α-helix content of the BSA as estimated by circular dichroism, and in a 21-fold decrease in the affinity of the α-amanitin moiety for calf thymus RNA polymerase II. The ID 50 values toward Chinese hamster ovary cells were 1.5 μ m , 3.0 μ m , and 4.7 μ m for ABGG-BSA, α-amanitin, and ABGG, respectively, indicating that conjugation of ABGG to BSA increased its inhibitory activity 3-fold over the unconjugated derivative. The conditions examined here define a potential with limitations for covalent conjugation via carbodiimide of amanitin to carrier proteins for targeting to mammalian cells.

The effect of a reporter molecule on chromatin template activity

Abstract A reporter molecule, which is said to bind exclusively to the minor groove of DNA, does not interfere with the transcription of chromatin by an exogenous E. coli RNA polymerase. This is in contrast to the marked inhibition of chromatin template activity by actinomycin D. Taken together with previous observations, these results support the hypothesis that the chromatin proteins regulating transcription by RNA polymerase are located in the major groove of DNA.

Topography of Nucleic Acid Helices in Solutions

Abstract A number of reporter molecules of the structure R-(CH2)n-N+(CH3)2(CH2)mN+(CH3)3·2Br−, where R is a chromophore absorbing in the 300–500 mp region, have been synthesized. The effect of DNA and RNA on the absorption, induced circular dichroism, and proton magnetic resonance spectra is reported. A red shift and a hypochromic effect on the absorption spectra of the bound chromophore is observed. In all cases where R is an “unsymmetrical” 4-nitroaniline chromophore, it is found that DNA and RNA induce an opposite CD in the absorption band of the bound reporter molecules. These results together with PMR studies are interpreted in terms of the structure of the nucleic acid systems in solutions.

Dissymmetric recognition of the helical sense of deoxyribonucleic acid and evidence for binding of reporter molecules from the minor groove of DNA

Abstract Through the utilization of optically active DNP-derivatives of l - and d -proline, evidence is presented which suggests that nucleic acids exist as right-handed helices in solution. The results of ultraviolet absorption, circular dichroism, proton magnetic resonance (pmr), T m of the helix-coil transition, viscometric, and binding studies are consistent with the above interpretation. It is shown that several types of DNA (i.e., salmon sperm, calf thymus, Micrococcus luteus , poly d(A-T)-poly d(A-T) and poly d(I-C)-poly d(I-C)) exist in a right-handed helical structure in solution. In addition, evidence is presented which strongly indicates that the 2,4-dinitroaniline ring of DNP-proline is intercalated between base-pairs of DNA and the prolyl side chain situated in the minor groove. Moreover, it is shown that the more sterically hindered DNP-derivatives exhibit a higher selectivity for A-T binding sites.