Nanometal surface energy transfer (NSET), which describes an energy transfer event from optically excited organic fluorophores to small metal nanoparticles, may be used as a molecular beacon/ruler similar to FRET, but with advantages over this classical technique. Here we use NSET to measure Mg(2+)-induced conformational changes for a hammerhead ribozyme and confirm these measurements using FRET. These optical experiments enhance our understanding of the different kinetic pathways for this ribozyme.

Cation-mediated RNA folding from extended to compact, biologically active conformations relies on a temporal balance of forces. The Mg2 +-mediated folding of the Tetrahymena thermophila ribozyme is characterized by rapid nonspecific collapse followed by tertiary-contact-induced compaction. This article focuses on an autonomously folding portion of the Tetrahymena ribozyme, its P4-P6 domain, in order to probe one facet of the rapid collapse: chain flexibility. The time evolution of P4-P6 folding was followed by global and local measures as a function of Mg2 + concentration. While all concentrations of Mg2 + studied are sufficient to screen the charge on the helices, the rates of compaction and tertiary contact formation diverge as the concentration of Mg2 + increases; collapse is greatly accelerated by Mg2 +, while tertiary contact formation is not. These studies highlight the importance of chain stiffness to RNA folding; at 10 mM Mg2 +, a stiff hinge limits the rate of P4-P6 folding. At higher magnesium concentrations, the rate-limiting step shifts from hinge bending to tertiary contact formati

The complex formed between the U2 and U6 small nuclear (sn)RNA molecules of the eukaryotic spliceosome plays a critical role in the catalysis of precursor mRNA splicing. Here, we have used enzymatic structure probing, 19 F NMR, and analytical ultracentrifugation techniques to characterize the fold of a protein-free biophysically tractable paired construct representing the human U2-U6 snRNA complex. Results from enzymatic probing and 19 F NMR for the complex in the absence of Mg 2+ are consistent with formation of a four-helix junction structure as a predominant conformation. However,19 F NMR data also identify a lesser fraction (up to 14% at 25°C) of a three-helix conformation. Based upon this distribution, the calculated ΔG for interconversion to the four-helix structure from the three-helix structure is approximately −4.6 kJ/mol. In the presence of 5 mM Mg 2+ , the fraction of the three-helix conformation increased to ∼17% and the Stokes radius, measured by analytical ultracentrifugation, decreased by 2%, suggesting a slight shift to an alternative conformation. NMR measurements demonstrated that addition of an intron fragment to the U2-U6 snRNA complex results in displacement of U6 snRNA from the region of Helix III immediately 5 ′ of the ACAGAGA sequence of U6 snRNA, which may facilitate binding of the segment of the intron adjacent to the 5 ′ splice site to the ACAGAGA sequence. Taken together, these observations indicate conformational heterogeneity in the protein-free human U2-U6 snRNA complex consistent with a model in which the RNA has sufficient conformational flexibility to facilitate inter-conversion between steps of splicing in situ.

Simple lysine conjugates are capable of selective DNA damage at sites approximating a variety of naturally occurring DNA-damage patterns. This process transforms single-strand DNA cleavage into double-strand cleavage with a potential impact on gene and cancer therapy or on the design of DNA constructs that require disassembly at a specific location. This study constitutes an example of DNA damage site recognition by molecules that are two orders of magnitude smaller than DNA-processing enzymes and presents a strategy for site-selective cleavage of single-strand nucleotides, which is based on their annealing with two shorter counterstrands designed to recreate the above duplex damage site.damage recognition ͉ double-stranded DNA cleavage ͉ phosphate recognition ͉ sequence-specific DNA modification A side from the common double helix, DNA forms a wide range of structural motifs, such as hairpin loops, triplex, tetraplex, and bulged structures, as well as nicks and gaps. The individual structural features of these motifs make them potential candidates for specific targeting (1-6). Among these structural elements, nicks and gaps are promising for the development of sequence-specific DNA cleavage, because they already feature a break in the phosphate backbone of DNA.Cleavage of the phosphate backbone of DNA can be caused by chemical reagents (7) such as radicals (8) and by radiation damage (9). To survive, cells develop enzymatic mechanisms for the repair that work efficiently on single-strand (ss) damage (10). Any further cleavage on the opposite strand at the damage site leads to ds cleavage, which is hard to repair (11). The ds cleavage requires either a bifunctional reagent (12-20) or detection and targeting of the damaged site. The only literature example of the latter is a complex natural antibiotic, bleomycin (21).In this paper, we show that simple lysine conjugates can identify ss damage sites with high selectivity and induce DNA cleavage at the strand opposite to the damage site (Scheme 1). Results and DiscussionWe used an internally 32 P-labeled ‡ 54-nt DNA (BW 54s, 5Ј-TAA TAC GAC TCA CTA TAG GCC CAG GGA AAA CTT GTA AAG GTC TAC CTA TCT *ATT; the 32 P label is indicated by an asterisk). This sequence incorporates several isolated guanine (G) nucleotides as well as GG diads, GGG triads, and an AT tract, the latter serving as a natural binding site for a family of lysine conjugates. We have shown that a combination of AT selectivity of binding and G selectivity of activation through photoinduced electron transfer (22-29) can be used for selective targeting of guanines flanking the AT tract (30).By annealing BW 54s with a variety of counterstrands, we built a family of constructs ( Fig. 1) inspired by a selection of sites that formed either in the process of chemical damage of DNA or, transiently, during enzymatic processing of DNA (31). Nicked DNA (constructs B and C) is involved in DNA topological transitions, DNA repair synthesis, and DNA replication of the lagging strand (32). Single-nucleotide gaps with 3Ј...

Since natural and synthetic photosensitizers can generate reactive intermediates on demand with a high degree of temporal and spatial control, they attract considerable interest as molecular tools for the modification of DNA. [1] Bis-tetrafluoropyridinyl (bis-TFP) enediynes are attractive organic DNA photocleavers because they do not require metals or reducing agents and because they are thermally and photochemically stable unless excited in the vicinity of a suitable electron donor such as DNA. Thus, not only light but also binding to DNA are necessary for the activation. Photoinduced electron transfer (PET) triggers transformation of these molecules into indenes (the C1-C5 cyclization, Scheme 1) with four concomitant formal H-atom abstractions.[2] In addition, bis-TFP enediynes can be excited with low-energy visible light under two-photon conditions, [3] the cyclization can be effectively controlled by substituents, [4] and, unlike the Bergman cyclization, [5, 6] individual steps in the C1-C5 cascade can lead to DNA photocleavage through either oxidative or hydrogen-abstraction mechanisms. Intriguingly, the nature of the reaction products suggests that reactivation of fulvene intermediates can lead to further DNA damage that develops after the cyclization step.In our recent work, we applied a supercoiled DNA relaxation assay to demonstrate that the ratio of doublestranded (ds) to single-stranded (ss) DNA photocleavage caused by lysine-enediyne conjugates 1 and 2 (Scheme 2) is greater than that expected for a series of random breaks. [8] The efficiency of the ds photocleavage compares favorably with DNA cleavage mediated by the natural enediyne calicheamicin g1 [9] and by bleomycin. [10,11] However, although this assay is a sensitive and convenient method for studies of new DNA photocleavers, it does not provide insight into the cleavage mechanism and sequence specificity of the DNA damage. Moreover, such important information is missing even in the case of more traditional enediyne photocleavers. In order to gain such an insight, we proceeded to investigate photoinduced damage of synthetic oligonucleotides by enediynes 1 and 2 equipped with a basic lysine moiety. These conjugates exhibit affinity towards DNA that is an order of magnitude greater [8] than that observed with spermine (29-36 mm), [12] a result suggesting that electrostatic binding of protonated amines to the negatively charged DNA backbone is complemented by noncovalent interactions with the enediyne moiety. 32 P-label indicated by asterisk) in aerated neutral phosphate buffer. This model 54-mer incorporates several isolated guanine (G) nucleotides as well as GG diads and GGG triads. Selective damage at these multiplet GGG or GG sites is a signature of PET from DNA, [13][14][15][16][17] which is the key step in the diversion of the reactivity of enediynes towards the C1-C5 pathway. Scheme 1. Comparison of photochemical Bergman (top) [5c, 7] and C1-C5(bottom) [2] cyclizations of enediynes. X = Y = H; X = Y = Me; X = H, Y = Cl; X = H, Y = NHCOR.Sc...