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Supporting information for Comolli et al. (2002) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.262663599

Ultraviolet absorbance and NMR melting experiments indicate that stem-loop "P2b" consists of two ordered domains in solution. Fig. 8 shows a side-by-side comparison between ultraviolet absorbance melting experiments (Fig. 8a), and 1D NMR temperature profiles (Fig. 8b).

The proton imino resonances from the stem of P2b give a very crowded region of H₂O-NOESY spectra, with few well-resolved cross-peaks (Fig. 9 a and b). The single adenine-uracil (AU) base-pair in the stem was first identified thanks to its sharp adenine H2 cross-peak with a characteristic AU downfield imino peak. The assignment of this peak in P2b_UUCG allowed us to establish clear connectivities from the stem to the loop, and from the AU base-pair towards the 5¢ end of the stem in D₂O-NOESY spectra (Fig. 10). These assignments proved useful in the assignment of the same type of connectivities for P2b.

One-dimensional NMR spectra collected on fully D₂O-exchanged samples show significant changes in P2b resonance peaks and in line widths when it is combined with either "P3" or "CR5" (Fig. 11).

A comparison between the proton imino region in H₂O-NOESY spectra of the complex "P2b:P3", and the complex between the loop domain of P2b and P3, "1/2P2b:P3", provides useful help in the assignments of the base-pairs for the pseudoknot in trans (Fig. 12). The central portion of the double-stranded RNA is readily assignable, but the AU base-pairs involving the three As at the 5’ end of P3 give no cross-peaks within the imino region. Two of them give cross-peaks to their own adenine H2 and water, proving that they form.

Very few proton imino resonances from the stem of P2b yield well-resolved cross-peaks. However, most imino resonances from the GC base-pairs give typical cross-peaks with the cytosines amino resonances and, in many cases, also with their H5 resonances. The current partial assignments were determined with the help of D₂O-NOESY and TOCSY spectra collected for all the constructs, and with a ¹⁵N-HMQC spectrum of a U-specific, ¹³C,¹⁵N-labeled P2b sample (Fig. 13). The U-specific labeled sample confirms that the first three pairs of Us in the P2b loop are involved in noncanonical base-pairs. The stem of P2b is closed by the G98C116 base-pair. Strong cross-peaks between the imino proton of G98 and the imino protons of U99 and U115 in H₂O-NOESY spectra connect the stem to the loop (Fig. 9a). The three noncanonical base-pairs formed by U99U115, U100U114, and U101U113 give two strong imino proton resonances each in H₂O-NOESY and ¹⁵N-HMQC spectra (Figs. 9a and 12). Strong cross-peaks establish a clear pattern of connectivities between all three UU base-pairs (Fig. 9a).

TOCSY spectra for P2b show the correct number of pyrimidine peaks, with some broadened resonances as expected from a molecule with this secondary structure (Fig. 14a). A comparison between the H5/H6 region of TOCSY spectra for free P2b and the complex "P2b:P3" shows a set of resonances in free P2b that completely disappear upon addition of P3, as a new set of resonances appears with the complex (Fig. 14 a and b). A comparison between the proton imino regions of H₂O-NOESY spectra of P2b and "P2b:CR5" shows that P2b does not loose its internal structure in the presence of CR5 (Fig. 15). The interaction is nonspecific, in the sense that it does not involve a well defined set of canonical base-pairs, as is the case for the complex P2b:P3.

Two- and one-dimensional proton NMR spectra of P2b and P2b-Dys sequences show a few specific differences between them (Figs. 16 and 17). This indicates different base-pairing, i.e., a difference in the secondary structure of the two sequences, and hence the different stability of the loops. When P2b dyskeratosis is combined with P3 wild-type, absolutely no changes are seen in the spectra (Fig. 17). The P2b loop does not loose its structure, and no base-pairing with P3 occurs.