e104 Nucleic Acids Research, 2008, Vol. 36, No. 16 PAGE 4 OF 8 Figure 1. Protocol and formation of DNA–STV–DNA dimers. (a) Schematic showing the two step incubation to optimize the eﬃciency of the dimer A+B, where A and B can be any nucleic acid with a biotin at one end. First, we bind molecule A to STV by 30 min incubation with a 30 molar excess of STV. We then remove all the unbound STV using gel puriﬁcation and incubate the STV-bound molecule A together with molecule B at 48C in a 1: 1 ratio for 30 min up to 15 h depending on the length of the molecules. (b) 0.8% agarose gel image showing the result of this protocol with 3.5 kb DNA (A) and 2.2 kb DNA (B) molecules (lane 1), and the control experiment without STV (lane 2). There is a very clear band at 5.7 kb (lane 1), corresponding to the dimer A + B. The eﬃciency of this reaction is 60%. (c) AFM image of the A+B heterodimer, and a height proﬁle traced along the molecule. The STV can be seen as a clear peak in the height proﬁle, and joins two molecules of about 1200 50 nm and 750 50 nm in length, corresponding well to the expected lengths for molecule A and B. bound STV reduces the electrophoretic mobility of DNA molecules up to at least 3.5 kb in length (Supplementary material). In the context of the second step of our reac- tion, the appearance of this band corresponding to A+STV may nonetheless appear somewhat surprising: for if STV is bound, why does dimer formation not follow and remove this band? To address this question, we have investigated whether the presence of this band could be decreased by longer incubation times or by vary- ing the relative concentrations of A and B; however, no observable eﬀect was found (Supplementary material). Consequently, we speculate that it is an imperfection in the STV that causes this band to remain: it is consistent with the existence of a fraction of STV molecules that can only bind single, but not multiple, biotins. Using the intensities of the bands at 3.7 kb (A+STV) and 5.7 kb (A+B), we can estimate the eﬃciency of for- mation of the dimer A+B and ﬁnd that to be 60% (see Materials and methods section). Note that in order to achieve such a high eﬃciency, it is necessary to minimize the formation of homodimers A+A and B+B (Supplementary material). We have employed two tech- nical steps to do so: ﬁrst, the gel extraction following the ﬁrst incubation ensures that all free STV is removed. Unfortunately, this is not the case for many other puriﬁ- cation methods, and small amounts of free STV can remain and will lead to the formation of B+B homodi- mers (Supplementary material). Second, the low tempera- ture in the second step prevents dissociation of the STV from the A+STV complex, which would otherwise also allow formation material). of homodimers (Supplementary Applications of the method End-joining of very long molecules. Having demonstrated that we can speciﬁcally form the heterodimers A+B with high eﬃciency, we now demonstrate the utility of this method in three applications: the end-joining of two very long DNA molecules, the end-joining of DNA to RNA, and the end-joining of RNA to RNA. In Figure 2a, lane 1, we demonstrate that we can eﬃciently end-join two very long molecules of DNA of 20 kb (A) and 9.6 kb (B). This is shown by the appearance of a band that migrates more slowly than the 23 kb marker in lane M2. This likely corresponds to the dimer A+B, which should migrate at approximately the same speed as 30 kb DNA. From the intensities of the bands, we deduce an eﬃciency of formation of the A+B dimer of 50%, similar to the case of the much shorter molecules in Figure 1. We therefore conclude that the eﬃciency does not signiﬁcantly decrease when joining substantially longer molecules. This is in sharp contrast to ligation, where the eﬃciency can decrease dramatically for such long molecules. This is illustrated in Figure 2b: Lane 1 shows the self-ligation of a 4.2 kb DNA molecule, resulting in an 8.4 kb DNA mole- cule. This 4.2 kb DNA molecule was digested with a restriction enzyme at a site in one of the PCR primers, leaving a 4 base pair self-complementary overhang (see Materials and methods section). After overnight