Thermodynamic and structural properties of pentamer DNA.cntdot.DNA, RNA.cntdot.RNA and DNA.cntdot.RNA duplexes of identical sequence

Abstract

Four pentamers with the general sequence 5'CU(T)GU(T)G/5'CACAG have been prepared by chemical synthesis in order to generate duplex structures with common sequences. The four duplexes studied include the DNA.DNA duplex (5'dCACAG/5'dCTGTG) and the RNA.RNA duplex (5'rCUGUG/5'rCACAG) as well as the two corresponding DNA.RNA heteroduplexes (5'rCUGUG/5'dCACAG and 5'CACAG/5'dCTGTG). The measured entropy, enthalpy, and free energy changes upon melting are reported for each pentamer and compared to the predicted values where possible. Results show that the two DNA.RNA heteroduplexes are destabilized (delta G degrees 25 = -4.2 +/- 0.4 kcal/mol) relative to either the DNA.DNA duplex (delta G degrees 25 = -4.8 +/- 0.5 kcal/mol) or the RNA.RNA duplex (delta G degrees 25 = -5.8 +/- 0.6 kcal/mol). Circular dichroism spectra indicate that the RNA and the two heteroduplexes adopt an A-form conformation, while the DNA conformation is B-form. Imino proton NMR spectra also show that the heteroduplex structures resemble the RNA.RNA duplex.

Degenerate target sites mediate rapid primed CRISPR adaptation

“…7B) and, surprisingly, these positions are all guanosines. This finding suggests that rG-dC (i.e., riboG-deoxyriboC) base pairing between the crRNA and the target DNA should not be disrupted for priming to occur, which is consistent with the fact that rG-dC base pairs are the strongest base pair known (32,33). The importance of rG is again reflected in the analysis of triple mutants (Fig.…”

Thermodynamic stability and solution conformation of tandem G · A mismatches in RNA and RNA · DNA hybrid duplexes

“…To compare the melting temperatures of the RNA duplexes with those of the analogous DNA duplexes, which were measured at 16 pM , we can use the values of A H and AS with Eqn (1) to calculate the T, values of the RNA duplexes at 16 pM. The Watson-Crick U(T)A duplexes have a T, that is 11 K higher in the RNA than in the DNA, in agreement with other studies (Hall and McLaughlin, 1991).…”

A single side chain prevents Escherichia coli DNA polymerase I (Klenow fragment) from incorporating ribonucleotides

“…The additional barriers that must be overcome when a DNA polymerase adds successive ribonucleotides could be of two general types: difficulty in accommodating a 2Ј-OH substituent on the primer-terminal residue at the active site, and problems with binding an RNA⅐DNA duplex in the primer-template (product) site. The preferred conformation for an RNA⅐DNA duplex is an A-like helix (32,33), whereas a DNA⅐DNA duplex, the normal product of a DNA polymerase, though favoring the B form, is able to assume either A-or B-form conformations. When DNA is bound to DNA polymerases the duplex tends to adopt a more A-like configuration close to the active site (6,7,(34)(35)(36), suggesting that a small stretch of ribonucleotides at the primer terminus might be tolerated reasonably well, but problems could arise if the newly synthesized RNA reached that part of the product binding site that is better suited for binding a B-form duplex.…”