Pyrimidine-rich Loop Research Articles

The 5'-terminal stem-loop RNA element of SARS-CoV-2 features highly dynamic structural elements that are sensitive to differences in cellular pH.

We present the nuclear magnetic resonance spectroscopy (NMR) solution structure of the 5'-terminal stem loop 5_SL1 (SL1) of the SARS-CoV-2 genome. SL1 contains two A-form helical elements and two regions with non-canonical structure, namely an apical pyrimidine-rich loop and an asymmetric internal loop with one and two nucleotides at the 5'- and 3'-terminal part of the sequence, respectively. The conformational ensemble representing the averaged solution structure of SL1 was validated using NMR residual dipolar coupling (RDC) and small-angle X-ray scattering (SAXS) data. We show that the internal loop is the major binding site for fragments of low molecular weight. This internal loop of SL1 can be stabilized by an A12-C28 interaction that promotes the transient formation of an A+•C base pair. As a consequence, the pKa of the internal loop adenosine A12 is shifted to 5.8, compared to a pKa of 3.63 of free adenosine. Furthermore, applying a recently developed pH-differential mutational profiling (PD-MaP) approach, we not only recapitulated our NMR findings of SL1 but also unveiled multiple sites potentially sensitive to pH across the 5'-UTR of SARS-CoV-2.

Advancing viral RNA structure prediction: measuring the thermodynamics of pyrimidine-rich internal loops.

Accurate thermodynamic parameters improve RNA structure predictions and thus accelerate understanding of RNA function and the identification of RNA drug binding sites. Many viral RNA structures, such as internal ribosome entry sites, have internal loops and bulges that are potential drug target sites. Current models used to predict internal loops are biased toward small, symmetric purine loops, and thus poorly predict asymmetric, pyrimidine-rich loops with >6 nucleotides (nt) that occur frequently in viral RNA. This article presents new thermodynamic data for 40 pyrimidine loops, many of which can form UU or protonated CC base pairs. Uracil and protonated cytosine base pairs stabilize asymmetric internal loops. Accurate prediction rules are presented that account for all thermodynamic measurements of RNA asymmetric internal loops. New loop initiation terms for loops with >6 nt are presented that do not follow previous assumptions that increasing asymmetry destabilizes loops. Since the last 2004 update, 126 new loops with asymmetry or sizes greater than 2 × 2 have been measured. These new measurements significantly deepen and diversify the thermodynamic database for RNA. These results will help better predict internal loops that are larger, pyrimidine-rich, and occur within viral structures such as internal ribosome entry sites.

High-throughput thermal stability assessment of DNA hairpins based on high resolution melting

On the basis of high-resolution melting, a high-throughput approach to measure melting temperatures (Tms) of short DNA hairpins was developed. With this method, Tms of thousands of triloop, tetraloop, and pentaloop hairpins involving various loop sequences and various closing base pairs (cbp) were obtained in hours. The stability of triloop hairpins decreased with the change of cbp (5′–3′) in the order of c-g > g-c > t-a ≥ a-t, showing that the cbp of 5′-Pyr-Pur-3′ (Pyr = pyrimidine, Pur = purine) contributed more stability than 5′-Pur-Pyr-3′. For tetraloop hairpins, GNNA, GNAB, and CNNG (N = A, G, C, or T; B = G, C, or T) were found to be highly stable irrespective of the cbp type. TNNA was also stable in both g-c and a-t families, while CGNA only in the c-g family. Pentaloop hairpins of cTGNAGg, cGNYNAg (Y = T or C) and cCGNNAg were exceptionally stable motifs. In most cases, pyrimidine-rich loops were more favorable to stabilize the whole structure than purine-rich ones. The present approach showed a good performance in assessing the thermal stability of large amounts of DNA hairpins comprehensively. These data are useful to understand the sequence dependence of the stability of DNA secondary structures and promising to improve the structure simulation by consummating basic databases.

Two-Dimensional Combinatorial Screening Identifies Specific Aminoglycoside−RNA Internal Loop Partners

Herein is described the identification of RNA internal loops that bind to derivatives of neomycin B, neamine, tobramycin, and kanamycin A. RNA loop-ligand partners were identified by a two-dimensional combinatorial screening (2DCS) platform that probes RNA and chemical spaces simultaneously. In 2DCS, an aminoglycoside library immobilized onto an agarose microarray was probed for binding to a 3 x 3 nucleotide RNA internal loop library (81,920 interactions probed in duplicate in a single experiment). RNAs that bound aminoglycosides were harvested from the array via gel excision. RNA internal loop preferences for three aminoglycosides were identified from statistical analysis of selected structures. This provides consensus RNA internal loops that bind these structures and include: loops with potential GA pairs for the neomycin derivative, loops with potential GG pairs for the tobramycin derivative, and pyrimidine-rich loops for the kanamycin A derivative. Results with the neamine derivative show that it binds a variety of loops, including loops that contain potential GA pairs that also recognize the neomycin B derivative. All studied selected internal loops are specific for the aminoglycoside that they were selected to bind. Specificity was quantified for 16 selected internal loops by studying their binding to each of the arrayed aminoglycosides. Specificities ranged from 2- to 80-fold with an average specificity of 20-fold. These studies show that 2DCS is a unique platform to probe RNA and chemical space simultaneously to identify specific RNA motif-ligand interactions.

Structural parameters affecting the kinetics of RNA hairpin formation

There is little experimental knowledge on the sequence dependent rate of hairpin formation in RNA. We have therefore designed RNA sequences that can fold into either of two mutually exclusive hairpins and have determined the ratio of folding of the two conformations, using structure probing. This folding ratio reflects their respective folding rates. Changing one of the two loop sequences from a purine- to a pyrimidine-rich loop did increase its folding rate, which corresponds well with similar observations in DNA hairpins. However, neither changing one of the loops from a regular non-GNRA tetra-loop into a stable GNRA tetra-loop, nor increasing the loop size from 4 to 6 nt did affect the folding rate. The folding kinetics of these RNAs have also been simulated with the program ‘Kinfold’. These simulations were in agreement with the experimental results if the additional stabilization energies for stable tetra-loops were not taken into account. Despite the high stability of the stable tetra-loops, they apparently do not affect folding kinetics of these RNA hairpins. These results show that it is possible to experimentally determine relative folding rates of hairpins and to use these data to improve the computer-assisted simulation of the folding kinetics of stem–loop structures.

Unusual DNA duplex and hairpin motifs.

Single-stranded DNA or double-stranded DNA has the potential to adopt a wide variety of unusual duplex and hairpin motifs in the presence (trans) or absence (cis) of ligands. Several principles for the formation of those unusual structures have been established through the observation of a number of recurring structural motifs associated with different sequences. These include: (i) internal loops of consecutive mismatches can occur in a B-DNA duplex when sheared base pairs are adjacent to each other to confer extensive cross- and intra-strand base stacking; (ii) interdigitated (zipper-like) duplex structures form instead when sheared G*A base pairs are separated by one or two pairs of purine*purine mismatches; (iii) stacking is not restricted to base, deoxyribose also exhibits the potential to do so; (iv) canonical G*C or A.T base pairs are flexible enough to exhibit considerable changes from the regular H-bonded conformation. The paired bases become stacked when bracketed by sheared G.A base pairs, or become extruded out and perpendicular to their neighboring bases in the presence of interacting drugs; (v) the purine-rich and pyrimidine-rich loop structures are notably different in nature. The purine-rich loops form compact triloop structures closed by a sheared G*A, A*A, A*C or sheared-like G(anti)*C(syn) base pair that is stacked by a single residue. On the other hand, the pyrimidine-rich loops with a thymidine in the first position exhibit no base pairing but are characterized by the folding of the thymidine residue into the minor groove to form a compact loop structure. Identification of such diverse duplex or hairpin motifs greatly enlarges the repertoire for unusual DNA structural formation.

Nucleolin associates with a subset of the human Ro ribonucleoprotein complexes.

Ro RNPs are evolutionarily conserved, small cytoplasmic RNA-protein complexes with an unknown function. In human cells, Ro RNPs consist of one of the four hY RNAs and two core proteins: Ro60 and La. Recently, the association of hnRNP I and hnRNP K with particles containing hY1 and hY3 RNAs has been described. The association of three other proteins, namely calreticulin, Ro52 and RoBPI, with (subsets of) the Ro RNPs is still controversial. To gain more insight into the composition and function of the Ro RNPs, we have immunopurified these particles from HeLa cell extracts using monoclonal antibodies against Ro60 and La. Using this approach, we have identified the RNA-binding protein nucleolin as a novel subunit of Ro RNP particles containing hY1 or hY3 RNA, but not hY4 and hY5 RNA. Using an in vitro hY RNA-binding assay we established that the internal pyrimidine-rich loop of hY1 and hY3 RNA is essential for the association of nucleolin. The binding is critically dependent on the presence of all four RNP motifs of nucleolin, but not of the C-terminal RGG-box. Moreover, we demonstrate that, in contrast to nucleolin and hnRNP K, nucleolin and hnRNP I can bind simultaneously to the internal pyrimidine-rich loop of hY1 RNA. We postulate that nucleolin functions in the biogenesis and/or trafficking of hY1 and hY3 RNPs through the nucleolus and subsequent transport to the cytoplasm.

Enhanced loop DNA folding induced by thymine-CH3 group contact and perpendicular guanine-thymine interaction.

A remarkable stabilizing effect induced by T-CH3 group and perpendicular guanine-thymine interactions in the DNA loop conformation has been demonstrated for the d(TTTG) loop structure using UV melting, high resolution NMR, distance geometry, and molecular dynamics studies. Contrary to the previously published d(TTCG) sequence that exhibits no specific inter-nucleotide interaction, we have found that d(TTTG), which differs only by one nucleotide with the d(TTCG) sequence (C7 --> T7), forms a rather stable and well-defined loop structure. Two characteristic structural features account for the stabilization of an otherwise flexible loop structure; the second loop T (T6) residue folds into the minor groove and engages in perpendicular interaction with the G8-NH2, while the third loop T (T7) residue stacks well upon the closing T5 x G8 wobble base pair and exhibits good contacts with many of the loop T5 and T6 sugar protons, which may form a hydrophobic core in the loop region. The importance of the bulky T7-CH3 was also proved by the UV melting study; while d(TTCG) hairpin exhibits a lower melting point (74.5 degrees C) than d(TTTG) hairpin (80.5 degrees C), d(TT(5-methyl)CG) hairpin resumes the same higher melting point (80 degrees C). Similarly, the fact that the melting temperature (74 degrees C) of d(TTTI) is lower than that of d(TTTG) indicates the critical role played by the G8-NH2 group. Our structural studies of the d(TTTG) loop indicate that DNA and RNA use a different strategy to establish stable tertiary folds. Comparison with several other pyrimidine-rich loop hairpins suggests that different minor-groove folding modes exist for the folding thymidine residue.

Stable formation of a pyrimidine-rich loop hairpin in a cruciform promoter

We have determined the solution structure of a TCC-loop hairpin in the cruciform promoter for the bacteriophage N4 virion RNA polymerase (N4 vRNAP). This hairpin and its complementary GGA-loop hairpin are extruded at physiological superhelical density and are required for vRNAP recognition. Contrary to its complementary GGA-loop, the three pyrimidines in the TCC-loop are all unpaired. However, with the help of two juxtaposed stem Watson-Crick G·C base-pairs, each nucleotide in the loop employs a special method to stabilize the hairpin structure. The resulting structures display extensive loop base-stacking rearrangement yet minor backbone distortion, which is largely accomplished through some loop ζ and α torsional angle changes. Consistent with the structural studies, UV melting of the GAAGCTCCGCTTC hairpin revealed a higher melting temperature (66 °C) than that of the GAACGTCCCGTTC hairpin (58 °C) with reversed stem G·C base-pairs, indicating significant contribution from the extra three loop-stem H-bonds. Thermodynamic parameters ΔG°25 of the GAAGCTCCGCTTC hairpin and its complementary GAAGCGGAGCTTC hairpin are −4.1 and −4.3 kcal/mol respectively, indicating approximately equal contribution of each hairpin to the cruciform formation of the N4 virion RNA polymerase promoter. No significant loop dynamics in the microsecond to millisecond NMR time-scale was observed, and the abundant well-defined exchangeable and non-exchangeable proton NOEs allowed us to efficiently determine a well-converged family for the final structures of the TCC-loop hairpin.

The loop E-loop D region of Escherichia coli 5S rRNA: the solution structure reveals an unusual loop that may be important for binding ribosomal proteins.

5S ribosomal RNA is the smallest rRNA. Its Watson-Crick helices were identified more than 20 years ago, but the conformations of its loops have long defied analysis. One of the three arms of 5S rRNA, residues 69-106 in Escherichia coli, contains a 14-residue internal loop called loop E. The sequence of loop E is conserved within kingdoms, and is terminated by a pyrimidine-rich loop called loop D. Loop E is the binding site for the ribosomal protein L25 in the E. coli ribosome. The solution structure of a 42-nucleotide derivative of E. coli 5S rRNA that includes loops D and E has been determined by nuclear magnetic resonance spectroscopy. Formally, loop E is not a loop at all; it is a double helical structure that contains seven, consecutive non-Watson-Crick base pairs. The major groove of the molecule is narrowed in loop E, and an unusual array of hydrogen-bond donors and acceptors appear in its minor groove. Loop D, which on paper looks like a three-pyrimidine terminal loop closed by a GC, is better thought of as a five-base loop because its closing GC is not a normal Watson-Crick pair. The two pyrimidines on the 5'-side of the loop are stacked on each other, and tilt into the minor groove of the adjacent helix. The third pyrimidine is fully exposed to solvent. This structure rationalizes all the biochemical and chemical protection data available for the loop E-loop D arm of intact 5S rRNA. While the molecule is double helical over its entire length, the geometry of its internal loop is highly irregular, and its irregularities may explain why the loop E-loop D arm of 5S rRNA interacts specifically with ribosomal protein L25 in E. coli.

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.

Nuclease resistance of an extraordinarily thermostable mini-hairpin DNA fragment, d(GCGAAGC) and its application to in vitro protein synthesis.

The nuclease resistance of a short, thermostable mini-hairpin, d(GCGAAGC), and other related hairpins was examined. Hairpins possessing a purine-rich (GAA) or (GAAA) loop appeared to be more resistant against nucleases than those with a pyrimidine-rich loop or single-stranded oligomers. Among 8 kinds of oligodeoxyribonucleotides examined, the fragment most resistant against nucleases was a hairpin with the sequence of d(CGCGAAGCG). This hairpin was then utilized for the stabilization of mRNA in an in vitro translation system; the 3'-terminal region of an mRNA was hybridized with an oligodeoxyribonucleotide including the sequence complementary to the 3'-terminus of the mRNA tagged with the nuclease-resistant d(CGCGAAGCG) hairpin sequence. By using this method, dihydrofolate reductase (DHFR) mRNA was stabilized against nucleases contaminating a cell-free translation system of E.coli, with a consequent increase in protein synthesis efficiency of 200%.

Strong binding of single-stranded DNA by stem-loop oligonucleotides.

We report that oligodeoxynucleotides which form stem-loop hairpin structures and which have pyrimidine-rich loops can form strong complexes with complementary single-stranded DNA sequences. Stem-loop oligonucleotides were constructed with a 25-nt T-rich loop and with variable Watson-Crick stems. The complexes of these oligomers with the sequence dA8 were studied by thermal denaturation. Evidence is presented that the complexes are one-to-one, bimolecular complexes in which the pyrimidine loop bases comprise the outer strands in a pyr.pur.pyr triplex, in effect chelating the purine strand in the center of the loop. Melting temperatures for the loop complexes are shown to be up to 29 degrees C higher than Watson-Crick duplex of the same length. It is shown that the presence of a stem increases stability of the triplex relative to an analogous oligomer without a stem. The effect of stem length on the stability of such a complex is examined. Such hairpin oligomers represent a new approach to the sequence-specific binding of single-stranded RNA and DNA. In addition, the finding raises the possibility that such a complex may exist in natural RNA folded sequences.