DNA-functionalized hydrogels for confined membrane-free in vitro transcription/translation.

We microfluidically fabricate bio-orthogonal DNA-functionalized porous hydrogels from hyaluronic acid that are employed in in vitro transcription/translation (IVTT) of a green fluorescent protein. By co-encapsulating individual hydrogel particles and the IVTT machinery in water-in-oil microdroplets, we study protein expression in a defined reaction volume. Our approach enables precise control over protein expression rates by gene dosage. We show that gene transcription and translation are confined to the membrane-free hydrogel matrix, which contributes to the design of membrane-free protocells.

Novel application of sRNA: stimulation of ribosomal frameshifting.

Small RNAs play an important role in regulation of gene expression in eukaryotic and eubacterial cells by modulating gene expression both at the level of transcription and translation. Here, we show that short complementary RNAs can also affect gene expression by stimulating ribosomal frameshifting in vitro. This finding has important implications for understanding the process of ribosomal frameshifting and for the potential application of small RNAs in the treatment of diseases that are due to frameshift mutations.

Solution structure of the pseudoknot of SRV-1 RNA, involved in ribosomal frameshifting.

RNA pseudoknots play important roles in many biological processes. In the simian retrovirus type-1 (SRV-1) a pseudoknot together with a heptanucleotide slippery sequence are responsible for programmed ribosomal frameshifting, a translational recoding mechanism used to control expression of the Gag-Pol polyprotein from overlapping gag and pol open reading frames. Here we present the three-dimensional structure of the SRV-1 pseudoknot determined by NMR. The structure has a classical H-type fold and forms a triple helix by interactions between loop 2 and the minor groove of stem 1 involving base-base and base-sugar interactions and a ribose zipper motif, not identified in pseudoknots so far. Further stabilization is provided by a stack of five adenine bases and a uracil in loop 2, enforcing a cytidine to bulge. The two stems of the pseudoknot stack upon each other, demonstrating that a pseudoknot without an intercalated base at the junction can induce efficient frameshifting. Results of mutagenesis data are explained in context with the present three-dimensional structure. The two base-pairs at the junction of stem 1 and 2 have a helical twist of approximately 49 degrees, allowing proper alignment and close approach of the three different strands at the junction. In addition to the overwound junction the structure is somewhat kinked between stem 1 and 2, assisting the single adenosine in spanning the major groove of stem 2. Geometrical models are presented that reveal the importance of the magnitude of the helical twist at the junction in determining the overall architecture of classical pseudoknots, in particular related to the opening of the minor groove of stem 1 and the orientation of stem 2, which determines the number of loop 1 nucleotides that span its major groove.

Structure of the ribozyme substrate hairpin of Neurospora VS RNA: a close look at the cleavage site.

The cleavage site of the Neurospora VS RNA ribozyme is located in a separate hairpin domain containing a hexanucleotide internal loop with an A-C mismatch and two adjacent G-A mismatches. The solution structure of the internal loop and helix la of the ribozyme substrate hairpin has been determined by nuclear magnetic resonance (NMR) spectroscopy. The 2 nt in the internal loop, flanking the cleavage site, a guanine and adenine, are involved in two sheared G.A base pairs similar to the magnesium ion-binding site of the hammerhead ribozyme. Adjacent to the tandem G.A base pairs, the adenine and cytidine, which are important for cleavage, form a noncanonical wobble A+-C base pair. The dynamic properties of the internal loop and details of the high-resolution structure support the view that the hairpin structure represents a ground state, which has to undergo a conformational change prior to cleavage. Results of chemical modification and mutagenesis data of the Neurospora VS RNA ribozyme can be explained in context with the present three-dimensional structure.

Structure of the 3'-hairpin of the TYMV pseudoknot: preformation in RNA folding.

The solution structure of an RNA-hairpin present in the pseudoknot, which is found at the 3'-terminus of turnip yellow mosaic virus genomic RNA, has been solved by nuclear magnetic resonance spectroscopy. The loop, which contains the sequence 5'-GGGUCA-3', was found to be highly structured and, contrary to expectations, does not attain its stability through GA or GC base pair formation but by triple interactions between the tilted adenosine and the minor groove sides of the first two guanosines. Interestingly, a very similar conformation was found for the cognate pseudoknot, implying that the 3'-hairpin is preformed for folding into a pseudoknotted structure. These findings suggest a mechanism of 'predetermined-fit' as a principle in RNA folding.

NMR structure of a classical pseudoknot: interplay of single- and double-stranded RNA.

Pseudoknot formation folds the 3' ends of many plant viral genomic RNAs into structures that resemble transfer RNA in global folding and in their reactivity to transfer RNA-specific proteins. The solution structure of the pseudoknotted T arm and acceptor arm of the transfer RNA-like structure of turnip yellow mosaic virus (TYMV) was determined by nuclear magnetic resonance (NMR) spectroscopy. The molecule is stabilized by the hairpin formed by the 5' end of the RNA, and by the intricate interactions related to the loops of the pseudoknot. Loop 1 spans the major groove of the helix with only two of its four nucleotides. Loop 2, which crosses the minor groove, interacts closely with its opposing helix, in particular through hydrogen bonds with a highly conserved adenine. The structure resulting from this interaction between the minor groove and single-stranded RNA at helical junctions displays internal mobility, which may be a general feature of RNA pseudoknots that regulates their interaction with proteins or other RNA molecules.

New developments in structure determination of pseudoknots.

Recently, several high-resolution structures of-RNA pseudoknots have become available. Here we review the progress in this area. The majority of the structures obtained belong to the classical or H-type pseudoknot family. The most complicated pseudoknot structure elucidated so far is the Hepatitis Delta Virus ribozyme, which forms a nested double pseudoknot. In particular, the structure-function relationships of the H-type pseudoknots involved in translational frameshifting have received much attention. All molecules considered show interesting new structural motifs.

The detailed structure of tandem G.A mismatched base-pair motifs in RNA duplexes is context dependent.

The solution structure of the RNA duplex (rGGGCUGAAGCCCU), containing tandem G.A mismatches has been determined by NMR spectroscopy and restrained molecular dynamics. A homonuclear 3D TOCSY-NOESY was used to derive 18 to 30 distance restraints per nucleotide, as well as all gamma torsion angles and sugar puckers for the central UGAA part of the molecule. Using these constraints, together with cross-strand distances, involving exchangeable imino protons, and essentially all other torsion angles that can accurately be determined (i.e. beta, epsilon) otherwise, the structure of the UGAA domain could be determined with high precision (r.m.s.d. 0.62 A), without the aid of isotopically enriched RNA. The G.A base-pairs are of the sheared pairing type, with both nucleotides in the anti conformation, and hydrogen bonds between the guanine 2-amino and the adenine N7 and between the guanine N3 and the adenine 6-amino. Surprisingly the sugar of the guanosine of the G.A. mismatch adopts a 2'-endo sugar pucker conformation. Comparison with other RNA structures, in which two such G.A base-pairs are formed reveals that this detailed structure depends on the identity of the base 5' to the guanosine in the tandem G.A base-pairs. A geometrical model for the incorporation of sheared tandem G.A base-pairs in A-form helices is formulated, which explains the distinct different stacking properties and helical parameters in sequences containing tandem, sheared G.A base-pairs.

The structure of the isolated, central hairpin of the HDV antigenomic ribozyme: novel structural features and similarity of the loop in the ribozyme and free in solution.

The structure of an RNA hairpin containing a seven-nucleotide loop that is present in the self-cleaving sequence of hepatitis delta virus antigenomic RNA was determined by high resolution NMR spectroscopy. The loop, which is composed of only one purine and six pyrimidines, has a suprisingly stable structure, mainly supported by sugar hydroxyl hydrogen bonds and base-base and base-phosphate stacking interactions. Compared with the structurally well-determined, seven-membered anticodon loop in tRNA, the sharp turn which affects the required 180 degrees change in direction of the sugar-phosphate backbone in the loop is shifted one nucleotide in the 3' direction. This change in direction can be characterized as a reversed U-turn. It is expected that the reversed U-turn may be found frequently in other molecules as well. There is evidence for a new non-Watson-Crick UC base pair formed between the first and the last residue in the loop, while most of the other bases in the loop are pointing outwards making them accessible to solvent. From chemical modification, mutational and photocrosslinking studies, a similar picture develops for the structure of the hairpin in the active ribozyme indicating that the loop structure in the isolated hairpin and in the ribozyme is very similar.

A network of heterogeneous hydrogen bonds in GNRA tetraloops.

RNA hairpin loops containing a GNRA consensus sequence are the most frequently occurring hairpins in a variety of prokaryotic and eukaryotic RNAs. These tetraloops play important functional roles in RNA folding, in RNA-RNA tertiary interactions and as protein binding sites. Homo and heteronuclear NMR spectroscopy have been used to determine the structures of the most abundant members of the GNRA tetraloop family: the GAGA, GCAA and GAAA loops closed by a C-G base pair. Analysis of the structures of these three hairpin loops reveals a network of heterogeneous hydrogen bonds. The loops contain a G-A base pair, a G base-phosphate hydrogen bond and several 2' OH-base hydrogen bonds. These intramolecular interactions and the extensive base stacking in the loop help explain the high thermodynamic stability and give insight into the diverse biological roles of the GNRA RNA hairpins.

Unambiguous structure characterization of a DNA-RNA triple helix by 15N- and 13C-filtered NOESY spectroscopy.

DNA.DNA*RNA triple helices of the pyrimidine.purine*pyrimidine motif (where . indicates Watson-Crick pairing and * indicates Hoogsteen pairing) appear to be very stable, which has important implications for the development of novel antisense strategies. Here we present the first structural NMR studies on such a system, composed of a DNA hairpin with a homopurine-homopyrimidine stem sequence and a single-stranded RNA oligonucleotide containing exclusively pyrimidine residues. In these investigations an unlabeled DNA hairpin and a uniformly 13C/15N-enriched RNA oligonucleotide were utilized in combination with X-edited 1H NMR spectroscopy. Improved 15N (omega 2) filtered NOESY and 13C (omega 1) filtered NOESY are presented by which we were able to differentiate between intrastrand, i.e., DNA-DNA and RNA-RNA, and interstrand, i.e., DNA-RNA, NOE contacts. It is unambiguously established that the complex forms a right-handed triple helix, with the RNA strand situated in the major groove of the Watson-Crick stem of the hairpin. The interaction is stabilized by the formation of Hoogsteen-type base pairs between the RNA strand and the purine strand of the DNA. These strands run parallel to each other. The characterization of the DNA-RNA triple helix structure described here shows that this type of experiment forms a valuable instrument in the structure determination of bimolecular systems of nucleic acids.

Irradiated (15N)DNA as an internal standard for analysis of base-oxidized DNA constituents by isotope dilution mass spectrometry.

A simple and convenient procedure for the preparation of isotopically labeled DNA enriched in oxidized deoxynucleosides is described. 15N-Labeled DNA was isolated from Escherichia coli cells grown in an isotopically enriched medium, and the level of oxidative damage was increased by in vitro irradiation under oxygen. The resulting DNA was hydrolyzed and subsequently analyzed by GC/MS. Results indicated that the DNA was 99% 15N-enriched and that 1% of the total 2'-deoxyguanosine was converted into 8-hydroxy-2'-deoxyguanosine (8-OHdG). When applied to the analysis of 8-OHdG, [15N]DNA as internal standard gave a better reproducibility (CV, 7.9%; n = 5) as compared to the monomeric 8-[18O]hydroxy-2'-deoxyguanosine (CV, 16%; n = 4). Background levels of 8-OHdG in rat colon DNA determined with [15N]DNA and 8-18OHdG as internal standard were 26 +/- 11 and 15 +/- 7 8-OHdG per 10(6) deoxynucleosides, respectively.

Sequential backbone assignment of uniformly 13C-labeled RNAs by a two-dimensional P(CC)H-TOCSY triple resonance NMR experiment.

A new 1H-13C-31P triple resonance experiment is described which allows unambiguous sequential backbone assignment in 13C-labeled oligonucleotides via through-bond coherence transfer from 31P via 13C to 1H. The approach employs INEPT to transfer coherence from 31P to 13C and homonuclear TOCSY to transfer the 13C coherence through the ribose ring, followed by 13C to 1H J-cross-polarisation. The efficiencies of the various possible transfer pathways are discussed. The most efficient route involves transfer of 31Pi coherence via C4'i and C4'i-1, because of the relatively large JPC4' couplings involved. Via the homonuclear and heteronuclear mixing periods, the C4'i and C4'i-1 coherences are subsequently transferred to, amongst others, H1'i and H1'i-1, respectively, leading to a 2D 1H-31P spectrum which allows a sequential assignment in the 31P-1H1' region of the spectrum, i.e. in the region where the proton resonances overlap least. The experiment is demonstrated on a 13C-labeled RNA hairpin with the sequence 5'(GGGC-CAAA-GCCU)3'.

Assignment strategies and analysis of cross-peak patterns and intensities in the three-dimensional homonuclear TOCSY-NOESY of RNA.

The application of 3D TOCSY-NOESY to the analysis of RNA is presented, using a TOCSY-NOESY spectrum of the RNA duplex r(5'GGGCUGAAGCCU'). It is shown that for RNA molecules, 3D spectra can be obtained with a digital resolution comparable to that obtained for 2D NMR with full spectral information. The improvement in assignment over 2D methods is shown and discussed on the basis of an assignment strategy presented earlier. A simple and straightforward method for determining sugar puckers and gamma backbone torsion angles is presented, which is derived from an analysis of cross-peak intensities originating from the TOCSY coherence transfer among sugar protons and H5'/5" protons. The stereospecific assignment of the H5'/5" resonances in 3D TOCSY-NOESY spectra is also discussed.

Structural features that give rise to the unusual stability of RNA hairpins containing GNRA loops.

The most frequently occurring RNA hairpins in 16S and 23S ribosomal RNA contain a tetranucleotide loop that has a GNRA consensus sequence. The solution structures of the GCAA and GAAA hairpins have been determined by nuclear magnetic resonance spectroscopy. Both loops contain an unusual G-A base pair between the first and last residue in the loop, a hydrogen bond between a G base and a phosphate, extensive base stacking, and a hydrogen bond between a sugar 2'-end OH and a base. These interactions explain the high stability of these hairpins and the sequence requirements for the variant and invariant nucleotides in the GNRA tetranucleotide loop family.

Nuclear magnetic resonance studies of the hammerhead ribozyme domain. Secondary structure formation and magnesium ion dependence.

Proton nuclear magnetic resonance (n.m.r.) experiments were used to probe base-pair formation in several hammerhead RNA enzyme (ribozyme) domains. The hammerhead domains consist of a 34 nucleotide ribozyme bound to a complementary 13 nucleotide non-cleavable DNA substrate. Three hammerhead domains were studied that differ in the sequence and stability of one of the helices involved in recognition of the substrate by the ribozyme. The n.m.r. data show a 1:1 stoichiometry for the ribozyme-substrate complexes. The imino proton resonances in the hammerhead complexes were assigned by two-dimensional nuclear Overhauser effect experiments. These data confirm the presence of two of the three helical regions in the hammerhead domain, predicted from phylogenetic data; and are also consistent with the formation of the third helix. Since a divalent cation is required for efficient catalytic activity of the hammerhead domain, the magnesium ion dependence of the n.m.r. spectra was studied for two of the hammerhead complexes. One of the complexes showed very large spectral changes upon addition of magnesium ions. However, the complex that has the most C.G base-pairs in one of the recognition helices shows essentially no spectral (and therefore presumably structural) changes upon addition of magnesium. These data are consistent with a model where the magnesium binding site already exists in the magnesium-free complex, suggesting that the magnesium ion serves primarily a catalytic, and not a structural, role under the conditions used here.

Is there a special function for U.G basepairs in ribosomal RNA?

U.G basepairs are well-established elements of RNA structure. The geometry of this pair is different, however, from classical Watson-Crick basepairs. This leads to an unusual stacking of the basepair: overlap with the basepair at the 5' side of the U (and the 3' side of the G) is strong (stacked) while it is weak with the basepair on the other side (destacked). The closure of an RNA helix by a U.G pair will be energetically unfavourable when the U residue occupies the 5' end. In transfer RNA there is a strong selection against a 'destacked' U.G pair at helix ends. In the 16S rRNA model of Escherichia coli there are 72 U.G pairs of which 36 or 22 occupy a helix end, depending on how such an end is defined. There is a slight preference for 'stacked' U.G's in these positions. It is remarkable, however, that of 13 very conserved U.G pairs in the 16S (-like) rRNA, 7 occur at helix ends and that 5 of these have the 'destacked' configuration. It is suggested that these pairs, if they exist at all in a hydrogen-bounded form, are stabilized by co-axial stacking with other helices or by interaction with a protein.

Conformational and thermodynamic effects of naturally occurring base methylations in a ribosomal RNA hairpin of Bacillus stearothermophilus.

The 3'-terminal colicin fragments of 16S ribosomal RNA were isolated from Bacillus stearothermophilus and from its kasugamycin-resistant (ksgA) derivative lacking N6-dimethylation of the two adjacent adenosines in a hairpin loop. The fragment from the ksgA strain still contains a naturally occurring N2-methylguanosine in the loop. An RNA molecule resembling the B. stearothermophilus colicin fragment but without modified nucleosides was synthesized in vitro using a DNA template and bacteriophage T7 RNA polymerase. Proton-NMR spectra of the RNAs were recorded at 500 MHz. The imino-proton resonances of base-paired G and U residues could be assigned on the basis of previous NMR studies of the colicin fragment of Escherichia coli and by a combination of methylation-induced shifts and thermal melting of base pairs. The assignments were partly confirmed by NOE measurements. Adenosine dimethylation in the loop has a distinct conformational effect on the base pairs adjoining the loop. The thermal denaturation melting curve of the enzymatically synthesized RNA fragment was also determined and the transition midpoint (tm) was found to be 73 degrees C at 15 mM Na+. A comparison with previously determined thermodynamic parameters for various colicin fragments demonstrates that base methylations in the loop lead to a relatively strong destabilization of the hairpin helix. In terms of free energy the positive contribution of the methylations are in the order of the deletion of one base pair from the stem. Other data show that recently published free-energy parameters do not apply for certain RNA hairpins.

Sequence-dependent structural variations of hammerhead RNA enzymes.

The discovery of in vivo catalytic activity for the hammerhead RNA self-cleaving domain has led to the development of a new class of sequence-specific RNA endonucleases. Two such ribozymes have been synthesized using in vitro transcription with T7 polymerase and their structures have been studied by optical spectroscopy, nuclear magnetic resonance and nondenaturing gel electrophoresis. These data show the presence of a stable hairpin consisting of a double helical stem and a tetranucleotide loop in both RNA enzymes. Additional structure, with different stabilities, is also observed in both RNA enzymes. The half-lives for cleavage of the complementary RNA substrates by these two RNA enzymes have been previously shown to differ by a factor of 50. The data presented here suggest that this rate difference may be a result of the formation of catalytically inactive conformations in the RNA enzyme which interfere with formation of the enzyme-substrate complex.