Pulsed field-gradient NMR experiments can be used to measure the diffusion constants of nucleic acids. The diffusion constants measured in this way for double-helical DNAs of defined length agree well both with theory and with measurements done using other techniques. When applied to RNAs, this experiment easily distinguishes duplex RNAs from RNA hairpins and thus it can solve one of the perennial problems faced by RNA spectroscopists, i.e. assessing whether their samples are monomeric or not.

The method of DNA cyclization kinetics reveals special properties of the TATAAACGCC sequence motif found in DNA sequences that have high affinity for core histones. Replacement of 30 bp of generic DNA by three 10-bp repeats of the motif in small cyclization constructs increases cyclization rates by two orders of magnitude. We document a 13°bend in the motif and characterize the direction of curvature. The bending force constant is smaller by nearly 2-fold and there is a 35% decrease in the twist modulus, relative to generic DNA. These features are the likely source of the high affinity for bending around core histones to form nucleosomes. Our results establish a protocol for determination of the ensembleaveraged global solution structure and mechanical properties of any Ϸ10-bp DNA sequence element of interest, providing information complementary to that from NMR and crystallographic structural studies.D NA in cells is highly compacted from the unfettered size it would have if free in solution. The first stage of packaging is achieved by wrapping DNA around core histones in a welldefined conformation (1), forming a beads-on-a-string structure that is further compacted by additional proteins into folded chromatin and ultimately into metaphase chromosomes. Interphase chromatin is generally less condensed, but it contains regulatory sites such as promoters at which DNA must be distorted to enable interactions between proteins that bind distant sites on linear DNA. As a relatively stiff polymer, DNA resists the bending required for packaging, which requires free energy assistance from strong interactions with core histones and other proteins.Variations in DNA sequence can, in principle, modulate the energy cost of bending and packaging. For example, intrinsic curvature of the kind provided by A-tracts (2) should, if in the proper direction, lessen the energy required for bending. Increased local bending flexibility could reduce the energetic cost of packaging, as could an increase in twist flexibility (3), assuming that the helical phasing between distal regulatory sequence elements must be altered to form a protein complex.The stiffness of DNA (the inverse of its flexibility) is traditionally described by the persistence length P, defined as the average projection of the end-to-end vector of a very long chain on its initial direction. Classical techniques, such as light scattering (4) and rotational diffusion (5), supplemented by DNA cyclization kinetics (6), have led to estimates of around 140-180 bp for P. However, these methods are generally unable to deconvolute rigorously the contributions of curvature and flexibility to apparent persistence length (7), unless special constructs are used (8).Parameter sets exist for predicting DNA curvature based on roll and tilt values for dinucleotide (9-13) and tetranucleotide (14) steps, but the dinucleotide parameters vary considerably from set to set (15). Nor is there sufficient data to define unambiguously the sequence dependence of the experimental persistence length, ...

Recent developments in multidimensional heteronuclear NMR spectroscopy and large-scale synthesis of uniformly 13C-and 15N-labeled oligonucleotides have greatly improved the prospects for determination of the solution structure of RNA. However, there are circumstances in which it may be advantageous to label only a segment of the entire RNA chain. For example, in a larger RNA molecule the structural question of interest may reside in a localized domain. Labeling only the corresponding nucleotides simplifies the spectrum and resonance assignments because one can filter proton spectra for coupling to 13C and '5N. Another example is in resolving alternative secondary structure models that are indistinguishable in imino proton connectivities. Here we report a general method for enzymatic synthesis of quantities of segmentally labeled RNA molecules required for NMR spectroscopy. We use the method to distinguish definitively two competing secondary structure models for the 5' half of Caenorhabditis elegans spliced leader RNA by compar- However, when dealing with RNAs larger than -35 nucleotides, the limitations of uniform labeling become apparent because of two fundamental problems. The first is associated with the twin difficulties of extensive spectral overlap due to the larger number of resonance and poorer intrinsic spectral resolution because of the increasing rotational correlation time. Many spectral editing techniques have been developed to simplify the spectra further (5).The second problem, which will be the focus of this paper, reflects the increased diversity of secondary structures that are accessible to longer and more complicated RNAs (6). ForThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.short oligonucleotides, such diversity is very limited and usually there is only one plausible secondary structure. For large oligonucleotides, however, there may be several possible secondary structures for one defined sequence. The problems caused by this diversity can be subdivided into two classes. First, multiple conformations of the RNA may exist, complicating the spectra and making their interpretation difficult. Therefore, for NMR studies, every effort is made in sample preparation to ensure that only one major conformation exists under the conditions studied. In the second class, only one major conformation exists but it may be difficult to define the correct secondary structure based on the exchangeable proton spectra. The difficulties in this second case arise from two independent sources-namely, spectral overlap, which is frequently encountered, and imino pathway degeneracy, which refers to the situation in which two distinct secondary structures have indistinguishable imino proton connectivities. If a defined sequence has two secondary structures that are degenerate in their imino pathway, there is no way to distinguish them based on a conv...

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