Fluorescence detection of single nucleotide polymorphisms using nucleic acid probe containing tricyclic base-linked acyclonucleoside

A reliable and simple method for detecting nucleobase mutations is very important clinically because sequence variations in human DNA cause genetic diseases and genetically influenced traits. A majority of sequence variations are attributed to single nucleotide polymorphisms (SNPs). Here, we developed a method for SNP detection using DNA probes that contained a fluorescent tricyclic base-linked acyclonucleoside N. The type of nucleobases involved in the SNP sites in an RNA target could be determined using four DNA probes containing N. Further, we found that the SNP in the RNA target could be detected by a visible color. Thus, this system would provide a novel and simple method for detecting SNPs in an RNA target.     

Rapid detection of alpha-1-antitrypsin deficiency by analysis of a PCR-induced TaqI restriction site

Summary A single base substitution is responsible for the PI-Z mutation in alpha-1-antitrypsin (AAT) deficiency. The Z mutation, which is in exon V of the AAT gene, was analysed directly using a primer designed with a single base substitution in the DNA sequence. During the polymerase chain reaction with this primer, a restriction enzyme site was created in the exon-V-amplified DNA sequence; this site was present in the normal allele (M form) but absent in the Z form. Here, the design of the primer and the application of the designer primer for prenatal diagnosis of chorion villus samples (CVS) for AAT deficiency is described. The method provides a simple rapid means of prenatal diagnosis of AAT deficiency within a day of the collection of the CVS. The detection of the nucleotide base change in AAT deficiency at the Z mutation site provides the opportunity for accurate prenatal diagnosis where no tissue is available from an AAT-affected individual.     

Sequence for human argininosuccinate synthetase cDNA.

The nucleotide sequence for human argininosuccinate synthetase cDNA was determined by analysis of six clones isolated from a single experiment. The sequence covered 1623 nucleotides including 76 bases of poly(A) and contained a 1236 nucleotide open reading frame encoding a protein of 46,434 daltons. In one cDNA isolate, a cloning artifact or perhaps RNA polymerase error involving addition of an A in a region of six A's within the coding sequence was documented. Single base variations in the 3' untranslated region were examined in detail since detection of DNA polymorphisms in the cDNAs could imply over-expression of both alleles at the active locus in canavanine-resistant cells, i.e. a trans-acting mechanism for enzyme overproduction. However, the sequence from five cDNAs suggested some single base artifacts, and DNA polymorphism remains uncertain. The occurrence of three tandem arginine codons in the 5' untranslated region of the cDNA suggested the possibility of an interaction of arginyl-tRNA with mRNA to regulate RNA processing or half-life as a mechanism for arginine-mediated repression.     

Protein-RNA sequence covariation in a ribosomal protein-rRNA complex

Comparative sequence analysis has successfully predicted secondary structure and tertiary interactions in ribosomal and other RNAs. Experiments presented here ask whether the scope of comparative sequence-based predictions can be extended to specific interactions between proteins and RNA, using as a system the well-characterized C-terminal RNA binding domain of ribosomal protein L11 (L11-C76) and its 58 nucleotide binding region in 23S rRNA. The surface of L11-C76 alpha-helix 3 is known to contact RNA; position 69 in this helix is conserved as serine in most organisms but varies to asparagine (all plastids) or glutamine (Mycoplasma). RNA sequence substitutions unique to these groups of organisms occur at base pairs 1062/1076 or 1058/1080, respectively. The possibility that rRNA base pair substitutions compensate for variants in L11 alpha-helix 3 has been tested by measuring binding affinities between sets of protein and RNA sequence variants. Stability of the RNA tertiary structure, as measured by UV melting experiments, was unexpectedly affected by a 1062/1076 base pair substitution; additional mutations were required to restore a stably folded structure to this RNA. The results show that the asparagine variant of L11-C76 residue 69 has been compensated by substitution of a 1062/1076 base pair, and plausibly suggest a direct contact between the amino acid and base pair. For some of the protein and RNA mutations studied, changes in binding affinity probably reflect longer-range adjustments of the protein-RNA contact surface.     

Targeting of A701G nucleotide at the human ATP1A1 locus using a RNA/DNA chimera

The single base substitution mediated by chimeric RNA/DNA oligonucleotide is a new promising approach of gene therapy for single base mutation diseases. We exploited this approach to render HeLa cells resistant to ouabain by introducing a single base substitution in the alpha 1 subunit of the NA+/K+ ATPase human gene. The chimeric oligonucleotide was administered to HeLa cells by electroporation and the frequency of ouabain resistant cells determined. The results showed that the chimeric RNA/DNA oligonucleotide failed to enhance the frequency of ouabain resistant cells supporting the controversy about the conflicting results of the technique.     

Complete nucleotide sequence of RNA 3 from alfalfa mosaic virus, strain S

We report the sequence of RNA 3 from strain S of Alfalfa mosaic virus (2,055 nucleotides). This RNA codes for a 32.4 kd protein (P3) and for the 24 kd coat protein (P4). The largest part of the sequence was established using RNA sequencing methods. The completion of the sequence in the region coding for P3 was achieved with cloned cDNA synthesized after priming at internal sites of RNA 3. Comparison of the RNA sequences coding P3 and P4 proteins in strain S with those reported in the literature for strain 425 revealed a higher amino acid substitution rate (3%) for P3 than for P4 (congruent to 1%) despite a similar average base substitution of 3-4% in these regions. In P3, two out of nine amino acid changes occur in hydrophilic regions. The amino acid changes in P4 do not modify the local hydrophilicity distribution. The intercistronic region displays a low degree of base substitution (2%) when compared with the untranslated 3'-end region (3.6%) or the 5'-end leader region (8%), the average substitution rate being 3.2%.     

Novel sequence variations in LAMA2 andSGCG genes modulating cis-acting regulatory elements and RNA secondary structure

In this study, we detected new sequence variations in LAMA2 and SGCG genes in 5 ethnic populations, and analysed their effect on enhancer composition and mRNA structure. PCR amplification and DNA sequencing were performed and followed by bioinformatics analyses using ESEfinder as well as MFOLD software. We found 3 novel sequence variations in the LAMA2 (c.3174+22_23insAT and c.6085 +12delA) and SGCG (c. (*) 102A/C) genes. These variations were present in 210 tested healthy controls from Tunisian, Moroccan, Algerian, Lebanese and French populations suggesting that they represent novel polymorphisms within LAMA2 and SGCG genes sequences. ESEfinder showed that the c. (*) 102A/C substitution created a new exon splicing enhancer in the 3'UTR of SGCG genes, whereas the c.6085 +12delA deletion was situated in the base pairing region between LAMA2 mRNA and the U1snRNA spliceosomal components. The RNA structure analyses showed that both variations modulated RNA secondary structure. Our results are suggestive of correlations between mRNA folding and the recruitment of spliceosomal components mediating splicing, including SR proteins. The contribution of common sequence variations to mRNA structural and functional diversity will contribute to a better study of gene expression.     

A candidate gene for human U1 RNA.

Clones containing sequences complementary to the small nuclear RNA U1 were isolated from the human DNA library of Lawn et al. (1978). Three clones were studied by hybridization and restriction enzyme cleavage. The results showed that the inserts in all three clones were different and that each clone contains one single copy of a sequence which hybridizes to U1 RNA. The results revealed moreover that only one of the three clones contains all the cleavage sites which can be predicted from the known sequence of human U1 RNA, suggesting that the three clones comprise one candidate U1 gene and two pseudogenes. A fragment from the recombinant with the candidate U1 gene was subcloned in the pPR322 plasmid and part of its sequence was determined. The results showed that the subclone contains a sequence which matches that of the human U1 RNA perfectly. The sequence "TATAT" which often is found adjacent to RNA polymerase II start sites, was identified 33-37 base pairs upstream from the beginning of the U1 sequence. Two ten base pairs long, nearly perfect, direct repeats were also identified in the vicinity of the U1 sequence and an imperfect inverted repeat follows immediately after the U1 gene.     

Optical spectroscopic study of the effects of a single deoxyribose substitution in a ribose backbone: implications in RNA-RNA interaction

The 2'-OH group in the ribose sugars of an RNA molecule plays an important role in guiding tertiary interactions that stabilize different RNA structural motifs. Deoxyribose, or 2'-OH by 2'-H, substitution in both the single-stranded and the duplex part of an RNA backbone has been routinely used to evaluate what role the 2'-OH plays in different tertiary interactions that guide an RNA-RNA contact. A deoxyribose substitution not only has the effect of removing a hydrogen bond donating group, but also introduces a sugar moiety with a preference for C2'-endo pucker in a backbone of predominantly C3'-endo sugars. This study evaluates the effects of a single deoxyribose substitution in both single-stranded and double-helical forms of RNA oligomers. A single-stranded, nonrepetitive 7-mer oligoribonucleotide (7-mer RNA) and four different variants having the same base sequence but with a single deoxyribose sugar at different positions in the strands have been studied by ultraviolet (UV) absorption, circular dichroism (CD), and Fourier transform infrared (FTIR) spectroscopy. Duplexes were formed by association with the complementary strand of the 7-mer RNA. The results show that both RNA and DNA single strands have preorganized conformations with spectral properties resembling those of A- and B-form helices, respectively, with RNA being more heterogeneous than its DNA counterpart. A single deoxyribose substitution perturbs the structure of the RNA backbone, with the effect being more pronounced in the single-stranded than in the duplex structure. The perturbation depends on the position of the 2'-H substitution in the strand.     

TARGETING OF A701G NUCLEOTIDE AT THE HUMAN ATP1A1 LOCUS USING A RNA/DNA CHIMERA

ABSTRACT

The single base substitution mediated by chimeric RNA/DNA oligonucleotide is a new promising approach of gene therapy for single base mutation diseases. We exploited this approach to render HeLa cells resistant to ouabain by introducing a single base substitution in the alpha 1 subunit of the NA⁺/K⁺ ATPase human gene. The chimeric oligonucleotide was administered to HeLa cells by electroporation and the frequency of ouabain resistant cells determined. The results showed that the chimeric RNA/DNA oligonucleotide failed to enhance the frequency of ouabain resistant cells supporting the controversy about the conflicting results of the technique.     

Evaluation of the use of S1 nuclease to detect small length variations in genomic DNA

A method which utilises S1 nuclease to detect small length variations in cloned and genomic DNA has been evaluated. The methodology of this technique is simple and robust, permitting the rapid analysis of 10(4) base pairs. By employing defined sequence variants, this method is shown to have a sensitivity which should enable the detection of length variations of only a few base pairs in heterozygous individuals.     

RNA-templated DNA ligation for transcript analysis

Ligase-mediated gene detection has proven valuable for detection and precise distinction of DNA sequence variants. We have recently shown that T4 DNA ligase can also be used to distinguish single nucleotide variants of RNA sequences. Here we describe parameters that influence RNA-templated DNA ligation by T4 DNA ligase. The reaction proceeds much more slowly, requiring more enzyme, compared to ligation of the same oligonucleotides hybridized to the corresponding DNA sequence. The reaction is inhibited at high concentrations of ATP and NaCl and both magnesium and manganese ions can support the reaction. We define reaction conditions where 80% of RNA target molecules can template a diagnostic ligation reaction. Ligase-mediated RNA detection should provide a useful mechanism for sensitive and accurate detection and distinction of RNA sequence variants.     

RSEARCH: Finding homologs of single structured RNA sequences

Background

For many RNA molecules, secondary structure rather than primary sequence is the evolutionarily conserved feature. No programs have yet been published that allow searching a sequence database for homologs of a single RNA molecule on the basis of secondary structure.

Results

We have developed a program, RSEARCH, that takes a single RNA sequence with its secondary structure and utilizes a local alignment algorithm to search a database for homologous RNAs. For this purpose, we have developed a series of base pair and single nucleotide substitution matrices for RNA sequences called RIBOSUM matrices. RSEARCH reports the statistical confidence for each hit as well as the structural alignment of the hit. We show several examples in which RSEARCH outperforms the primary sequence search programs BLAST and SSEARCH. The primary drawback of the program is that it is slow. The C code for RSEARCH is freely available from our lab's website.

Conclusion

RSEARCH outperforms primary sequence programs in finding homologs of structured RNA sequences.

     

The tobacco mosaic virus assembly origin RNA. Functional characteristics defined by directed mutagenesis

The in vitro reassembly of tobacco mosaic virus (TMV) begins with the specific recognition by the viral coat protein disk aggregate of an internal TMV RNA sequence, known as the assembly origin (Oa). This RNA sequence contains a putative stem-loop structure (loop 1), believed to be the target for disk binding in assembly initiation, which has the characteristic sequence AAGAAGUCG exposed as a single strand at its apex. We show that a 75-base RNA sequence encompassing loop 1 is sufficient to direct the encapsidation by TMV coat protein disks of a heterologous RNA fragment. This RNA sequence and structure, which is sufficient to elicit TMV assembly in vitro, was explored by site-directed mutagenesis. Structure analysis of the RNA identified mutations that appear to effect assembly via a perturbation in RNA structure, rather than by a direct effect on coat protein binding. The binding of the loop 1 apex RNA sequence to coat protein disks was shown to be due primarily to its regularly repeated G residues. Sequences such as (UUG)3 and (GUG)3 are equally effective at initiating assembly, indicating that the other bases are less functionally constrained. However, substitution of the sequences (CCG)3, (CUG)3 or (UCG)3 reduced the assembly initiation rate, indicating that C residues are unfavourable for assembly. Two additional RNA sequences within the 75-base Oa sequence, both of the form (NNG)3, may play subsidiary roles in disk binding. RNA structure plays an important part in permitting selective protein-RNA recognition, since altering the RNA folding close to the apex of the loop 1 stem reduces the rate of disk binding, as does shortening the stem itself. Whereas the RNA sequence making up the hairpin does not in general affect the specificity of the protein-RNA interaction, it is required to present the apex signal sequence in a special conformation. Mechanisms for this are discussed.     

Effects of site-specific substitution of 5-fluorouridine on the stabilities of duplex DNA and RNA.

The effects of 5-fluorouridine (FUrd) and 5-fluorodeoxyuridine (FdUrd) substitution on the stabilities of duplex RNA and DNA have been studied to determine how FUrd substitution in nucleic acids may alter the efficiency of biochemical processes that require complementary base pairing for molecular recognition. The parent sequence, 5'-GCGAAUUCGC, contains two non-equivalent uridines. Eight oligonucleotides (four RNA and four DNA) were prepared with either zero, one or two Urd substituted by FUrd. The stability of each self-complementary duplex was determined by measuring the absorbance at 260 nm as a function of temperature. Tm values were calculated from the first derivative of the absorbance versus temperature profiles and values for delta H0 and delta S0 were calculated from the concentration dependence of the Tm. Individual absorbance versus temperature curves were also analyzed by a parametric approach to calculate thermodynamic parameters for the duplex to single-stranded transition. Analysis of the thermodynamic parameters for each oligonucleotide revealed that FUrd substitution had sequence-dependent effects in both A-form RNA and B-form DNA duplexes. Conservation of helix geometry in FUrd-substituted duplexes was determined by CD spectroscopy. FUrd substitution at a single site in RNA stabilized the duplex (delta delta G37 = 0.8 kcal/mol), largely due to more favorable stacking interactions. FdUrd substitution at a single site in DNA destabilized the duplex (delta delta G37 = 0.3 kcal/mol) as a consequence of less favorable stacking interactions. All duplexes melt via single cooperative transitions.     

A review on architecture of the gag-pol ribosomal frameshifting RNA in human immunodeficiency virus: a variability survey of virus genotypes

Programmed ‘-1’ ribosomal frameshifting is necessary for expressing the pol gene overlapped from a gag of human immunodeficiency virus. A viral RNA structure that requires base pairing across the overlapping sequence region suggests a mechanism of regulating ribosome and helicase traffic during expression. To get precise roles of an element around the frameshift site, a review on architecture of the frameshifting RNA is performed in combination of reported information with augments of a representative set of 19 viral samples. In spite of a different length for the viral RNAs, a canonical comparison on the element sequence allocation is performed for viewing variability associations between virus genotypes. Additionally, recent and historical insights recognized in frameshifting regulation are looked back as for indel and single nucleotide polymorphism of RNA. As specially noted, structural changes at a frameshift site, the spacer sequence, and a three-helix junction element, as well as two Watson–Crick base pairs near a bulge and a C–G pair close a loop, are the most vital strategies for the virus frameshifting regulations. All of structural changes, which are dependent upon specific sequence variations, facilitate an elucidation about the RNA element conformation-dependent mechanism for frameshifting. These facts on disrupting base pair interactions also allow solving the problem of competition between ribosome and helicase on a same RNA template, common to single-stranded RNA viruses. In a broad perspective, each new insight of frameshifting regulation in the competition systems introduced by the RNA element construct changes will offer a compelling target for antiviral therapy.