e104 Nucleic Acids Research, 2008, Vol. 36, No. 16 STV to crosslink DNA molecules has been performed, this has primarily focused on the coupling of oligonucleotides or relatively short DNA molecules (27–31) up to 2 kb (32). Also, such research typically had the aim of either forming multimers of DNA–STV complexes or long strings of bis- biotinylated molecules (biotin on both ends) for the pur- pose of scaﬀolding in nanoengineering (30,31,33) or as useful aids in detection assays (29,34). Methods that speciﬁcally aim to optimize the formation of a unique end-product, such as a heterodimer A+B, are lacking. STV is an excellent choice as a coupling agent, because it binds biotin with an extremely high binding aﬃnity (Kd 10 15 M) and has multiple biotin binding sites (35,36). Indeed, wild-type STV contains four binding sites for biotin, so it could in principle join up to four biotin-labeled molecules. By simply adding STV to a col- lection of diﬀerent biotinylated molecules, one would therefore expect to form a variety of dimers, trimers and tetramers. To speciﬁcally promote the formation of the heterodimer A+B, we here introduce a two step approach, in which the STV is ﬁrst bound only to mole- cule A. Molecule B is then added only after the remaining unbound STV is removed, so that B must bind to the STV attached to molecule A. This not only avoids the forma- tion of homodimers A+A and B+B, but, as we show below, also signiﬁcantly suppresses the formation of tri- mers and tetramers. MATERIALS AND METHODS Synthesis of DNA and RNA DNA molecules were synthesized by PCR derived from phage lambda DNA (Promega). For all DNA molecules used to bind STV, only one of the primers was 50-biotiny- lated, so that only one end of each molecule can bind STV. The following forward and reverse primers (Biolegio, Leiden, The Netherlands) were used for the fragments: 2.2 kb (2250 bp) DNA: 50-CCAGAAAAATGCATTCCG TG-30 (forward) and 50-biotin-GGATATTAATACTGA AACTGAGATCAAGC-30 (reverse). 3.5 kb (3524 bp) DNA: 50-AAAAGAATTCAGCCTCAGCTGACCAGC CAGAAAACGACC-30 (forward) and 50-biotin- CTTG TTGGGCTTGTTTAATCCAGTAACTGC-30 (reverse). 4.2 kb (4215 bp) DNA: 50-TGATATTGCCAAAACAGA GCTG (forward) and 50-GGAAAGGGCCCGTAAAGT GATAATGATTATCATC-30 (reverse). The underlined sequence corresponds to a restriction site for ApaI. 9.6 kb (9571 bp) DNA: 50-AAAAAAGCTTGCGAGAA TTTTTAGCCCAAGC-30 (forward) and 50-biotin-GG ATATTAATACTGAAACTGAGATCAAGC (reverse). 12.7 kb (12668 bp) DNA: 50-biotin-GAGGCCGGGTTA TTCTTGTTCTCTGG (forward) and 50-GGAAAGGG CCCGTAAAGTGATAATGATTATCATC-30 (reverse). 20 kb (19945 bp) DNA: 50-biotin-GACGCAGGGGACC TGCAG (forward) and 50-AAAAGGTCTCTTCATGC GTTCAGTCTTAAAAGCAATT (reverse). The 4.2 kb and 12.7 kb DNA molecules were used in the ligation reaction in Figure 2b. The reverse primer is iden- tical for both molecules and includes an ApaI site PAGE 2 OF 8 (underlined). The molecules were digested overnight with an excess of ApaI at 258C. After digestion, both molecules contain the same overhang for ligation. The ligation reac- tions were performed in equimolar concentrations in a 20 ml volume with T4 DNA ligase (New England Biolabs, Ipswich, MA, USA) in T4 ligase buﬀer. All PCR fragments and digested molecules were puriﬁed with the nucleospin extract II kit (Machery-Nagel, Du¨ ren, Germany) and concentrations were determined using the commercial instrument Nanodrop (Isogen, IJsselstein, The Netherlands). The 4.2 kb single-stranded RNA (ssRNA) and double- stranded RNA (dsRNA) molecules used in Figure 3a were synthesized via run-oﬀ transcription of DNA PCR frag- ments derived from pBADb10 (a recombinant pBAD vector) using T7 RNA polymerase. The PCR primers (Biolegio, The Netherlands) for the template DNA were designed to incorporate a T7 RNA promoter sequence at one end of the PCR fragment. The template DNA for the ssRNA molecule contained the following forward and reverse primers (the T7 promoter is underlined): 50-TA ATACGACTCACTATAGGAAGATTAGCGGATCCT ACCTGAC-30 (forward) and 50-CGCAGCCAGCCAT CGGAACCGGGGTTAACCTCAACTTCC-30 (reverse). A small biotinylated oligonucleotide, 50-bio-CGCAGC CAGCCAUCGGAACC-30, was hybridized to the 50-end of the 4.2 kb ssRNA molecule to facilitate the binding to STV. For the dsRNA molecule, the complementary strand of the same DNA template was also transcribed by introdu- cing the T7 promoter in the reverse primer. In this case, the PCR primers used for the DNA template were: 50-A AGATTAGCGGATCCTACCTGAC-30 (forward) and 50-TAATACGACTCACTATAGGGGTTAACCTCAAC TTCCATTTCC-30 (reverse). Run-oﬀ transcription of this DNA template yields an ssRNA molecule that is complementary to and slightly shorter than the 4.2 kb ssRNA molecule described above. After hybridization of these two RNA molecules, the double-stranded product includes a 20 nt 30 overhang, to which the same biotinylated oligonucleotide was hybridized as used above. All hybridizations were per- formed following a procedure described previously (37). DNA–STV and RNA–STV binding All incubations were performed in buﬀer 1 (0.25 TBE at pH 8.3, 2 mM MgCl2). Unless stated otherwise, the ﬁrst incubation step was always performed at 378C for a dura- tion of 30 min, and the second incubation step was per- formed at 48C for 30 min up to several hours. Removal of STV We used two diﬀerent methods to remove unbound STV following the ﬁrst incubation step: column puriﬁcation using the Microcon YM-100 (Millipore, Amsterdam, The Netherlands), and gel puriﬁcation. The column pur- iﬁcation was carried out by loading the sample onto the column and adding ddH2O up to a total volume of 200 ml. After 1 min of centrifugation at 10 000 rpm, 200 ml ddH2O was added, and the sample was again centrifuged