Structural and Functional Characterization of Potent Antithrombotic Oligonucleotides Possessing Both Quadruplex and Duplex Motifs

Abstract

We report the results of a selection for single-stranded DNA oligonucleotide ligands to the serine protease thrombin using recently developed methods. This selection yielded a family of DNA sequences that conform to a consensus structure comprised of a unimolecular quadruplex motif and complementary flanking sequences capable of forming an additional Watson-Crick duplex motif. This novel quadruplex/duplex structure was not reported in a previous selection for DNA molecules which bind to thrombin [Bock et al. (1992) Nature 355, 564-566]. All quadruplex/duplex molecules tested bound to thrombin with higher affinity than quadruplex structures lacking the duplex structure. However, binding affinities did not always correlate with inhibitory potency since some molecules with high affinity were not potent inhibitors in vitro. 1H NMR spectroscopy studies demonstrated that the complementarity of bases in the duplex portion of a selected sequence allows it to form multimolecular structures. Constraining these molecules to the unimolecular quadruplex/duplex structure by bridging the 5' and 3' ends of the duplex motif with either triethylene glycol or disulfide bonds improved their thrombin inhibitory activity. All bridged quadruplex/duplex molecules were more potent inhibitors than molecules with only a quadruplex motif. Bridging the ends of these structures not only increased thrombin inhibition but also improved resistance to nucleases in serum more than 40-fold over the unbridged quadruplex. In addition, we have found that both the length and sequence of the duplex motif are important for inhibition.

Molecular recognition elements - DNA/RNA-aptamers to proteins

“…3). В ряде других лабораторий также была проведена селекция к тромбину, используя метод SELEX [36,37]. 15ТВА влияет и на агрегацию тромбоцитов, стимулированную тромбином.…”

Comparative termodynamic analysis of thrombin interaction with anti-thrombin aptamers and their heterodimeric construct

“…При длинах линкера от 35 до 55 нт, средние значения константы диссоциации комплексов тромбина с конструкциями достигали минимума (K D =0,2-0,4 нМ) и были приблизительно в 30 раз меньше, чем для исходных аптамеров. Гетеродимерные конструкции состоят из двух аптамеров, которые, как считается, селективно взаимодействуют с экзосайтами тромбина, расположенными приблизительно на противоположных сторонах белковой молекулы [23][24][25]. Усиление аффинности может определяться тем, что при определённых длинах линкера становится возможным формирование двух контактов между молекулой тромбина и гетеродимерной конструкцией [14].…”

“…Аптамер А2 содержит дополнительно фланкирующие последовательности, способные образовывать дуплекс (подчёркнуты) по аналогии с антитромбиновыми ДНК-аптамерами, полученными Macaya и соавт. [25]. Гетеродимерную аптамерную конструкцию А1(35)А2 получали, синтезируя олигонуклеотиды, состоящие из аптамерных последовательностей А1 и А2 (считая с 5'-конца), соединённых поли-(dT)-линкером протяжённостью 35 нт.…”

Aptamer-based therapeutics and their potential in radiopharmaceutical design

“…The exception to the rule is found in G-quadruplexes such as the DNA thrombin aptamer that seems to hold its structure even in the absence of its partner. The sampling of "shape space" in SELEX libraries has proven effective for the isolation of ligands to targets that are not known to interact with nucleic acids physiologically (Macaya et al, 1995). The known fact that proteins possess extensive surfaces with ridges, grooves, projections, and depressions, all covered with numerous H-bond donors and acceptors, suggests that proteins should be excellent targets for aptamer selections.…”