Potent 2'-Amino-2'-deoxypyrimidine RNA Inhibitors of Basic Fibroblast Growth Factor

Abstract

Screening of random oligonucleotide libraries with SELEX [systematic evolution of ligands by exponential enrichment; Tuerk, C., & Gold, L. (1990) Science 249, 505-510] has emerged as a powerful method for identifying high-affinity nucleic acid ligands for a wide range of molecular targets. Nuclease sensitivity of unmodified RNA and DNA, however, imposes considerable restrictions on their use as therapeutics or diagnostics. Modified RNA in which pyrimidine 2'-hydroxy groups have been substituted with 2'-amino groups (2'-aminopyrimidine RNA) is known to be substantially more resistant to serum nucleases. We report here on the use of SELEX to identify high-affinity 2'-aminopyrimidine RNA ligands to a potent angiogenic factor, basic fibroblast growth factor (bFGF). High-affinity ligands with the same consensus primary structure have been isolated from two independent libraries of approximately 6 x 10(14) molecules containing 30 or 50 randomized positions. Compared to unmodified RNA with the same sequence, 2'-aminopyrimidine ligands are at least 1000-fold more stable in 90% human serum. The sequence information required for high-affinity binding to bFGF is contained within 24-26 nucleotides. The minimal ligand m21A (5'-GGUGUGUGGAAGACAGCGGGUGGUUC-3'; G = guanosine, A = adenosine, C = 2'-amino-2'-deoxycytidine, U = 2'-amino-2'-deoxyuridine, and C = 2'-amino-2'-deoxycytidine or deoxycytidine) binds to bFGF with an apparent dissociation constant (Kd) of 3.5 +/- 0.3) x 10(-10) M at 37 degrees C in phosphate-buffered saline (pH 7.4). Disassociation of m21A from bFGF is adequately described with a first-order rate constant of (1.96 +/- 0.08) x 10(-3) s-1 (t1/2 = 5.9 min). The calculated value for the association rate constant (kon = k(off)/Kd) was 5.6 x 10(6) M-1 s-1. Highly specific binding of m21A to bFGF was observed: binding to denatured bFGF, five proteins from the FGF family (acidic FGF, FGF-4, FGF-5, FGF-6, and FGF-7), and four other heparin binding proteins is substantially weaker under the same conditions with KdbFGF/Kdprotein values ranging from (4.1 +/- 1.4) x 10(-2) to > 10(-6). Heparin but not chondroitin sulfate competed for binding of m21A to bFGF. In cell culture, m21A inhibited [125I]bFGF binding to both low-affinity sites (ED50 approximately 1 nM) and high-affinity sites (ED50 approximately 3 nM) on CHO cells expressing transfected FGF receptor-1.(ABSTRACT TRUNCATED AT 400 WORDS)

Aptamers That Preferentially Bind Type IVB Pili and Inhibit Human Monocytic-Cell Invasion by Salmonella enterica Serovar Typhi

“…Being smaller than antibodies, aptamers are better candidates for cell penetration and blood clearance. A variety of chemical modifications, such as fluorescent probes, cross-linking reagents, and modifications to the backbone or specific bases by fluorine (2Ј-hydroxyl groups of the ribose moieties are replaced with fluorine) (7), can be introduced, thereby adding stability and functionality. Moreover, aptamers are nonimmunogenic and therefore do not cause side effects resulting from unwanted immune responses in hosts.…”

Bioactive and nuclease-resistant l -DNA ligand of vasopressin

“…Selectionreflection thus joins the approach of using modified RNA polymerase substrates (11)(12)(13)(14) as a viable means of coupling the power of in vitro selection to the production of stable bioactive ligands.…”

Molecular recognition elements - DNA/RNA-aptamers to proteins

“…Селекция РНК к FIХa была проведена Rusconi C.P. с сотрудниками, и после восьми раундов селекции был найден аптамер, который связывался с FIХa с К d = 0,65±0,2 пМ и обладал 5000-кратным сродством к FIХa по сравнению с факторами FVIIa, FХa, FХIa и активированным протеином С [20]. Укороченная версия аптамера к FIХa (9.3t) сохраняла высокое сродство к FIХa (К d = 0,58±0,1 пМ) и полностью блокировала гидролиз FХ энзимным комплексом.…”

“…С помощью метода SELEX были выполнены селекционные циклы против VEGF 165 [19,20,[53][54][55] с использованием 2`-фторзамещенных пиримидинов в РНК библиотеке, Ruckman J. провел 12 раундов селекции и выделил аптамеры с К d = 50 пМ. Для увеличения стабильности против нуклеаз два аптамера дополнительно модифицировали по 2`-О-положению [55].…”

“…Для того чтобы защитить молекулы от действия неспецифических нуклеаз, при селекции используют пиримидиновые нуклеотиды, модифицированные по 2`-позициям рибозы (аминопроизводные и фторпроизводные) [18][19][20], используют липосомы в качестве носителя [21], проводят постселекционную модификацию гидроксильного радикала по 2`-положению введением метильных, аллильных, аминогрупп и т.д. [22][23][24].…”