Ion Selective Folding of Loop Domains in a Potent Anti-HIV Oligonucleotide

Abstract

Previously, we have described inhibition of HIV-1 infection by T30177, 5'-(GTGGTGGGTGGGTGGGT)-3', an oligonucleotide that is a potent inhibitor of HIV-1 integrase in vitro (Mazumder et al. (1996) Biochemistry 35, 13762). Here a family of oligonucleotides, analogs of T30177, has been studied. On the basis of thermal denaturation, we show that a folded structure of T30177 is much more stable than that of the thrombin binding aptamer, which only differs with T30177 in the loop sequence. Sequence changes reveal that loop interactions are solely responsible for this observed stability difference. In the presence of K+ ion, the fold of T30695, a designed 16mer derivative, is indeed more stable than T30177. Loop folding within T30695 is very ion selective. Quantitative analysis of thermal denaturation suggests that the loops of T30695, 5'-(GGGTGGGTGGGTGGGT)-3', and T30177 confer the ability to coordinate three equivalents of K+ ion (one bound to the core octet and two bound to the loops); however, the thrombin binding aptamer is shown to bind only one K+ equivalent. Folding kinetics and CD titration demonstrate that K+-induced folding of T30695 and T30177 is a two-step process, consistent with a sequential model in which a first equivalent of K+ binds to the octet core, followed by slow K+-induced rearrangement of the loop domains. Comparing structural stability with the capacity of the folded oligomers to inhibit the HIV-1 integrase enzyme in vitro or HIV-1 infection in cell culture, we have found that the folding and activity data are highly correlated, suggesting that formation of an orderly, ion-coordinated loop structure similar to that in T30177 or T30695 may be a prerequisite for both integrase inhibition and anti-HIV-1 activity.

Direct detection of aptamer-thrombin binding via surface-enhanced Raman spectroscopy

“…͔; cancer biomarkers; [22][23][24][25] HIVassociated proteins; [26][27][28][29] food-borne pathogens; 30 and other biologically important biomolecules such as insulin, 31,32 immunoglobulin E ͑IgE͒, 33 and thrombin. 34 They exhibit protein binding affinities that are comparable to those of corresponding antibodies.…”

Investigation of formation, recognition, stabilization, and conversion of dimeric G-quadruplexes of HIV-1 integrase inhibitors by electrospray ionization mass spectrometry

“…The inhibitory activities of the G-quadruplexes are related to their properties [11,12], such as stability and conversion. In this research, the base sequences of single-stranded oligonucleotides are 5=-GGGTGGGT-GGGTGGGT-3= (S1) and (5=-GTGGTGGGTGGGT-GGGT-3= (S2), two inhibitors of potent nanomolar HIV-1 integrase [9 -14].…”

Structure-Activity of Tetrad-forming Oligonucleotides as a Potent Anti-HIV Therapeutic Drug

“…Based upon T30177, a 16-residue oligonucleotide, T30695, 5Ј-g*ggtgggtgggtggg*t, was designed in order to improve both the structural stability and the inhibition of HIV-1 integrase activity (11). Thermal denaturation analysis demonstrated that T30695 folded into an extremely stable intramolecular G-quartet structure in the presence of K ϩ .…”