The influence of locked nucleic acid residues on the thermodynamic properties of 2′-O-methyl RNA/RNA heteroduplexes

The influence of locked nucleic acid (LNA) residues on the thermodynamic properties of 2′-O-methyl RNA/RNA heteroduplexes is reported. Optical melting studies indicate that LNA incorporated into an otherwise 2′-O-methyl RNA oligonucleotide usually, but not always, enhances the stabilities of complementary duplexes formed with RNA. Several trends are apparent, including: (i) a 3′ terminal U LNA and 5′ terminal LNAs are less stabilizing than interior and other 3′ terminal LNAs; (ii) most of the stability enhancement is achieved when LNA nucleotides are separated by at least one 2′-O-methyl nucleotide; and (iii) the effects of LNA substitutions are approximately additive when the LNA nucleotides are separated by at least one 2′-O-methyl nucleotide. An equation is proposed to approximate the stabilities of complementary duplexes formed with RNA when at least one 2′-O-methyl nucleotide separates LNA nucleotides. The sequence dependence of 2′-O-methyl RNA/RNA duplexes appears to be similar to that of RNA/RNA duplexes, and preliminary nearest-neighbor free energy increments at 37°C are presented for 2′-O-methyl RNA/RNA duplexes. Internal mismatches with LNA nucleotides significantly destabilize duplexes with RNA

A chemical synthesis of LNA-2,6-diaminopurine riboside, and the influence of 2′-O-methyl-2,6-diaminopurine and LNA-2,6-diaminopurine ribosides on the thermodynamic properties of 2′-O-methyl RNA/RNA heteroduplexes

Modified nucleotides are useful tools to study the structures, biological functions and chemical and thermodynamic stabilities of nucleic acids. Derivatives of 2,6-diaminopurine riboside (D) are one type of modified nucleotide. The presence of an additional amino group at position 2 relative to adenine results in formation of a third hydrogen bond when interacting with uridine. New method for chemical synthesis of protected 3′-O-phosphoramidite of LNA-2,6-diaminopurine riboside is described. The derivatives of 2′-O-methyl-2,6-diaminopurine and LNA-2,6-diaminopurine ribosides were used to prepare complete 2′-O-methyl RNA and LNA-2′-O-methyl RNA chimeric oligonucleotides to pair with RNA oligonucleotides. Thermodynamic stabilities of these duplexes demonstrated that replacement of a single internal 2′-O-methyladenosine with 2′-O-methyl-2,6-diaminopurine riboside (DM) or LNA-2,6-diaminopurine riboside (DL) increases the thermodynamic stability (ΔΔG°37) on average by 0.9 and 2.3 kcal/mol, respectively. Moreover, the results fit a nearest neighbor model for predicting duplex stability at 37°C. D-A and D-G but not D-C mismatches formed by DM or DL generally destabilize 2′-O-methyl RNA/RNA and LNA-2′-O-methyl RNA/RNA duplexes relative to the same type of mismatches formed by 2′-O-methyladenosine and LNA-adenosine, respectively. The enhanced thermodynamic stability of fully complementary duplexes and decreased thermodynamic stability of some mismatched duplexes are useful for many RNA studies, including those involving microarrays

Isoenergetic penta- and hexanucleotide microarray probing and chemical mapping provide a secondary structure model for an RNA element orchestrating R2 retrotransposon protein function

LNA (locked nucleic acids, i.e. oligonucleotides with a methyl bridge between the 2′ oxygen and 4′ carbon of ribose) and 2,6-diaminopurine were incorporated into 2′-O-methyl RNA pentamer and hexamer probes to make a microarray that binds unpaired RNA approximately isoenergetically. That is, binding is roughly independent of target sequence if target is unfolded. The isoenergetic binding and short probe length simplify interpretation of binding to a structured RNA to provide insight into target RNA secondary structure. Microarray binding and chemical mapping were used to probe the secondary structure of a 323 nt segment of the 5′ coding region of the R2 retrotransposon from Bombyx mori (R2Bm 5′ RNA). This R2Bm 5′ RNA orchestrates functioning of the R2 protein responsible for cleaving the second strand of DNA during insertion of the R2 sequence into the genome. The experimental results were used as constraints in a free energy minimization algorithm to provide an initial model for the secondary structure of the R2Bm 5′ RNA

Atomic resolution structure of CAG RNA repeats: structural insights and implications for the trinucleotide repeat expansion diseases

CAG repeats occur predominantly in the coding regions of human genes, which suggests their functional importance. In some genes, these sequences can undergo pathogenic expansions leading to neurodegenerative polyglutamine (poly-Q) diseases. The mutant transcripts containing expanded CAG repeats possibly contribute to pathogenesis in addition to the well-known pathogenic effects of mutant proteins. We have analysed two crystal forms of RNA duplexes containing CAG repeats: (GGCAGCAGCC)2. One of the structures has been determined at atomic resolution (0.95 Å) and the other at 1.9 Å. The duplexes include non-canonical A–A pairs that fit remarkably well within a regular A-helix. All the adenosines are in the anti-conformation and the only interaction within each A–A pair is a single C2-H2···N1 hydrogen bond. Both adenosines in each A–A pair are shifted towards the major groove, although to different extents; the A which is the H-bond donor stands out more (the ‘thumbs-up’ conformation). The main effect on the helix conformation is a local unwinding. The CAG repeats and the previously examined CUG structures share a similar pattern of electrostatic charge distribution in the minor groove, which could explain their affinity for the pathogenesis-related MBNL1 protein

The 3′ Splice Site of Influenza A Segment 7 mRNA Can Exist in Two Conformations: A Pseudoknot and a Hairpin

The 3′ splice site of influenza A segment 7 is used to produce mRNA for the M2 ion-channel protein, which is critical to the formation of viable influenza virions. Native gel analysis, enzymatic/chemical structure probing, and oligonucleotide binding studies of a 63 nt fragment, containing the 3′ splice site, key residues of an SF2/ASF splicing factor binding site, and a polypyrimidine tract, provide evidence for an equilibrium between pseudoknot and hairpin structures. This equilibrium is sensitive to multivalent cations, and can be forced towards the pseudoknot by addition of 5 mM cobalt hexammine. In the two conformations, the splice site and other functional elements exist in very different structural environments. In particular, the splice site is sequestered in the middle of a double helix in the pseudoknot conformation, while in the hairpin it resides in a two-by-two nucleotide internal loop. The results suggest that segment 7 mRNA splicing can be controlled by a conformational switch that exposes or hides the splice site

Revision of AMBER Torsional Parameters for RNA Improves Free Energy Predictions for Tetramer Duplexes with GC and iGiC Base Pairs

All-atom force fields are important for predicting thermodynamic, structural, and dynamic properties of RNA. In this paper, results are reported for thermodynamic integration calculations of free energy differences of duplex formation when CG pairs in the RNA duplexes r(CCGG)2, r(GGCC)2, r(GCGC)2, and r(CGCG)2 are replaced by isocytidine–isoguanosine (iCiG) pairs. Agreement with experiment was improved when ε/ζ, α/γ, β, and χ torsional parameters in the AMBER99 force field were revised on the basis of quantum mechanical calculations. The revised force field, AMBER99TOR, brings free energy difference predictions to within 1.3, 1.4, 2.3, and 2.6 kcal/mol at 300 K, respectively, compared to experimental results for the thermodynamic cycles of CCGG → iCiCiGiG, GGCC → iGiGiCiC, GCGC → iGiCiGiC, and CGCG → iCiGiCiG. In contrast, unmodified AMBER99 predictions for GGCC → iGiGiCiC and GCGC → iGiCiGiC differ from experiment by 11.7 and 12.6 kcal/mol, respectively. In order to test the dynamic stability of the above duplexes with AMBER99TOR, four individual 50 ns molecular dynamics (MD) simulations in explicit solvent were run. All except r(CCGG)2 retained A-form conformation for ≥82% of the time. This is consistent with NMR spectra of r(iGiGiCiC)2, which reveal an A-form conformation. In MD simulations, r(CCGG)2 retained A-form conformation 52% of the time, suggesting that its terminal base pairs may fray. The results indicate that revised backbone parameters improve predictions of RNA properties and that comparisons to measured sequence dependent thermodynamics provide useful benchmarks for testing force fields and computational methods

Synthesis of Oligoribonucleotides Containing N6‐Alkyladenosine and 2‐Methylthio‐N6‐Alkyladenosine

The N6‐alkyladenosines and 2‐methylthio‐N6‐alkyladenosines are the most common modified adenosine nucleosides, and transfer ribonucleic acids (tRNA) are particularly rich in these modified nucleosides. They are present at position 37 of the anticodon arm, and the contributions of these hypermodified nucleosides to codon‐anticodon interactions as well as to translation are significant, although they are not fully understood. This unit describes a new chemical synthesis method for oligoribonucleotides containing N6‐alkyladenosines and 2‐methylthio‐N6‐alkyladenosines via postsynthetic modifications of precursor oligoribonucleotides. To obtain oligoribonucleotides containing N6‐alkyladenosines, a precursor oligoribonucleotide carrying 6‐methylthiopurine riboside residues was used, whereas for the synthesis of oligoribonucleotides containing 2‐methylthio‐N6‐alkyladenosines, a precursor oligoribonucleotide carrying the 2‐methylthio‐6‐chloropurine riboside was applied. This allowed synthesis of modified oligoribonucleotides containing naturally occurring modified nucleosides such as N6‐isopentenyladenosine (i6A), N6‐methyladenosine (m6A), 2‐methylthio‐N6‐isopentenyladenosine (ms2i6A), and 2‐methylthio‐N6‐methyladenosine (ms2m6A), as well as several unnaturally modified adenosine derivatives.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/153079/1/cpnc0423.pd