Branched RNAs with two and four strands were synthesized. These structures were used to obtain branched siRNA. The branched siRNA duplexes had similar inhibitory capacity as those of unmodified siRNA duplexes, as deduced from gene silencing experiments of the TNF-α protein. Branched RNAs are considered novel structures for siRNA technology, and they provide an innovative tool for specific gene inhibition. As the method described here is compatible with most RNA modifications described to date, these compounds may be further functionalized to obtain more potent siRNA derivatives and can be attached to suitable delivery systems. 1. Introduction In recent years, siRNAs have generated tremendous interest in therapeutics [1]. Nevertheless, the transition of siRNAs from the laboratory to the clinical practice has encountered several obstacles. Briefly, siRNA duplexes are rapidly degraded in serum by exonucleases and endonucleases [2]. The polyanionic phosphodiester backbone of siRNA suffers from difficult cell uptake [3], and oligonucleotides may have off-target effects, either by stimulating the immune system [4] or by entering other endogenous gene regulation pathways [5]. Several chemical modifications have been proposed in the literature to address these drawbacks [2–4]. Most of these modifications are based on modified nucleosides and changes on backbone linkages [6, 7]. Thus, changes in sugar moiety influences sugar conformation, and, therefore, overall siRNA structure. Modifications of the 2′-OH by F or OMe as well as LNA [8, 9] are well tolerated and improve binding affinity and nuclease resistance. Base modifications that stabilize base pairs (5-bromouracil, 5-methylcytosine, 5-propynyluracil, and others) have also been proposed [7, 10]. Terminal conjugates, especially at the termini of the sense strand, have been modified with a large number of lipids to achieve improved cellular uptake [11]. In addition to these modifications, siRNA architecture is also crucial in the design of effective and specific siRNA. The architecture itself can be altered by chemical synthesis. In addition to the canonical siRNA architecture of 21-nt antiparallel, double-strand RNA with 2-nt 3′-overhangs [12], several forms of siRNA have been described. Blunt-ended siRNA [13], 25/27？mer Dicer-substrate or asymmetric siRNA [14] are among the siRNA structures formed by two strands. Moreover, functional siRNA can also be formed by one single RNA strand. This is the case in small hairpin RNA (shRNA), where the two strands are linked by a single loop [15], or RNA dumbbells [16],
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