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.

Solution structure of a HNA-RNA hybrid.

BACKGROUND: Synthetic nucleic acid analogues with a conformationally restricted sugar-phosphate backbone are widely used in antisense strategies for biomedical and biochemical applications. The modified backbone protects the oligonucleotides against degradation within the living cell, which allows them to form stable duplexes with sequences in target mRNAs with the aim of arresting their translation. The biologically most active antisense oligonucleotides also trigger cleavage of the target RNA through activation of endogenous RNase H. Systematic studies of synthetic oligonucleotides have also been conducted to delineate the origin of the chirality of DNA and RNA that are both composed of D-nucleosides. RESULTS: Hexitol nucleic acids (HNA) are the first example of oligonucleotides with a six-membered carbohydrate moiety that can bind strongly and selectively to complementary RNA oligomers. We present the first high resolution nuclear magnetic resonance structure of a HNA oligomer bound to a complementary RNA strand. The HNA-RNA complex forms an anti-parallel heteroduplex and adopts a helical conformation that belongs to the A-type family. Possibly, due to the rigidity of the rigid chair conformation of the six-membered ring both the HNA and RNA strand in the duplex are well defined. The observed absence of end-fraying effects also indicate a reduced conformational flexibility of the HNA-RNA duplex compared to canonical dsRNA or an RNA-DNA duplex. CONCLUSIONS: The P-P distance across the minor groove, which is close to A-form, and the rigid conformation of the HNA-RNA complex, explain its resistance towards degradation by Rnase H. The A-form character of the HNA-RNA duplex and the reduced flexibility of the HNA strand is possibly responsible for the stereoselectivity of HNA templates in non-enzymatic replication of oligonucleotides, supporting the theory that nucleosides with six-membered rings could have existed at some stage in molecular evolution.

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.

A physical and transcriptional map of the preaxial polydactyly locus on chromosome 7q36.

Preaxial polydactyly is a congenital hand malformation that includes duplicated thumbs, various forms of triphalangeal thumbs, and duplications of the index finger. A locus for preaxial polydactyly has been mapped to a region of 1.9 cM on chromosome 7q36 between polymorphic markers D7S550 and D7S2423. We constructed a detailed physical map of the preaxial polydactyly candidate region. With a combination of methods we identified and positioned 11 transcripts within this map. By recombination analysis on families with preaxial polydactyly, using newly developed polymorphic markers, we were able to reduce the candidate region to approximately 450 kb. The homeobox gene HLXB9, a putative receptor C7orf2, and two transcripts of unknown function, C7orf3 and C7orf4, map in the refined candidate region and have been subjected to mutation analysis in individuals with preaxial polydactyly.

Clinical and genetic studies on 12 preaxial polydactyly families and refinement of the localisation of the gene responsible to a 1.9 cM region on chromosome 7q36.

Polydactyly is the most frequently observed congenital hand malformation with a prevalence between 5 and 19 per 10000 live births. It can occur as an isolated disorder, in association with other hand/foot malformations, or as a part of a syndrome, and is usually inherited as an autosomal dominant trait. According to its anatomical location, polydactyly can be generally subdivided into pre- and postaxial forms. Recently, a gene responsible for preaxial polydactyly types II and III, as well as complex polysyndactyly, has been localised to chromosome 7q36. In order to facilitate the search for the underlying genetic defect, we ascertained 12 additional families of different ethnic origin affected with preaxial polydactyly. Eleven of the kindreds investigated could be linked to chromosome 7q36, enabling us to refine the critical region for the preaxial polydactyly gene to a region of 1.9 cM. Our findings also indicate that radial and tibial dysplasia/aplasia can be associated with preaxial polydactyly on chromosome 7q36. Combining our results with other studies suggests that all non-syndromic preaxial polydactylies associated with triphalangism of the thumb are caused by a single genetic locus, but that there is genetic heterogeneity for preaxial polydactyly associated with duplications of biphalangeal thumbs. Comparison of the phenotypic and genetic findings of different forms of preaxial polydactyly is an important step in analysing and understanding the aetiology and pathogenesis of these limb malformations.

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.

Genetics of limb development and congenital hand malformations.

The vertebrate limb bud develops along three different axes: proximodistal, anteroposterior, and dorsoventral. Several genetic factors responsible for control of each of the three limb axes have been identified. The genes involved interact in complex feedback loops to achieve proper arrangement and differentiation of tissues. Most of the available information on limb development and patterning has come from studies carried out in the lower vertebrates. In recent years, an increasing number of studies have been unraveling the genetic basis of human hand malformation phenotypes. At present, genes responsible for preaxial polydactyly, split hand/split foot malformation, and brachydactyly type C have been localized, and the gene responsible for synpolydactyly has been identified. In this paper, we present an overview of the genetic factors involved in limb development, followed by summarized discoveries in the genetics of human congenital hand malformations.

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.