PAGE 5 OF 8 Nucleic Acids Research, 2008, Vol. 36, No. 16 e104 Figure 2. End-joining very long DNA. (a) Pulsed ﬁeld electrophoresis gel image illustrating the eﬃcient end-joining of two very long DNA molecules. Lane 1 contains the product after joining a 20 kb (A) DNA molecule to a 9.6 kb (B) DNA molecule using our two step protocol. Using markers M1 and M2 as a reference, we can see bands corre- sponding to the molecules A, B, and an additional band corresponding to DNA longer than 23 kb, which is likely the A + B dimer of 30 kb DNA. The eﬃciency of dimer formation ( 50%) is similar to that of the shorter molecules in Figure 1. (b) Pulsed ﬁeld electrophoresis image illustrating that the ligation eﬃciency to join two molecules is strongly reduced for longer molecules: In lane 1 we show the ligation of a 4.2 kb long DNA molecule to itself (the molecules have a small self- complementary overhang), which clearly has a very high eﬃciency of about 90%, judging from the relative intensity of the band at about 8.5 kb DNA. However, the ligation of a 12.7 kb molecule with exactly the same overhang is much less eﬃcient: a band at 25 kb is not observed. incubation of this molecule with T4 DNA ligase, almost all molecules are ligated (lane 1). However, when the same reaction was carried out with a 12.7 kb molecule contain- ing exactly the same self-complementary overhang, the eﬃciency of the ligated product was dramatically reduced, to almost zero (lane 2). Note that in these two ligation reactions the molar concentrations of both mole- cules were kept the same for the long and the short mole- cules. Eﬀorts to improve the eﬃciency of the ligation reaction by performing the same reaction in the presence of poly-ethylene glycol (41), did not have noticeable eﬀects (results not shown). Further optimization may yield a higher eﬃciency, but this may be a time-consuming task. Conveniently, our biotin–STV linkage procedure oﬀers a way to join such molecules without the need for further optimization. End-joining of DNA to RNA. A second advantage of using STV as a linker is its versatility: for instance, it allows us to very straightforwardly join RNA molecules to DNA molecules. Normally, this would require the synthesis of complementary overhangs on the DNA and RNA molecules, and possibly the subsequent ligation of the RNA–DNA hybrid. This can be challenging, because secondary structure in the single-stranded DNA parts and RNA can prevent eﬃcient hybridization. In addition, DNA–RNA hybrid ligation reactions can be very ineﬃ- cient (42,43). In contrast, our biotin–STV linkage proce- dure only requires that both molecules are biotinylated at one end. Figure 3. End-joining DNA to RNA and RNA to RNA. (a) Joining DNA to RNA. Lane 1 contains the result following our two step protocol without addition of STV, using a 3.5 kb DNA (A) molecule and a 4.2 kb ssRNA molecule (B). The ssRNA is seen to migrate at the approximately at the same speed as 1.5 kb DNA and –as expected- we see only bands corresponding to molecules A and B. In lane 2, where the protocol was followed in the presence of STV, a band appears at 5 kb DNA, corresponding to the A + B dimer. In lanes 3 and 4 we illustrate the same procedure with two diﬀerent molecules: a 2.2 kb DNA molecule (A) and a 4.2 kb dsRNA molecule (B). Again, in the control experiment without STV no dimers are formed (lane 4). Conversely, in lane 3, after following our regular protocol in the pres- ence of STV, an additional band is observed at 6.5 kb. As the bare dsRNA molecule migrates at 4.2 kb DNA, the band at 6.5 kb most likely corresponds to the A + B dimer. The eﬃciencies in both cases are similar to or even better than DNA–DNA binding shown in Figure 1. (b) Joining RNA to RNA. The same 4.2 kb dsRNA molecule used above was used with our protocol to create an 8.4 kb dsRNA molecule. Lane 1 contains the result following our two-step protocol without addition of STV, using the 4.2 kb RNA molecule as both A and B: no dimers are formed. However, in the presence of STV (lane 2) an additional band is formed at 8.5 kb DNA corresponding to the dsRNA–dsRNA dimer. In Figure 3a we show two examples in which we bind DNA molecules to single-stranded and double-stranded RNA molecules, respectively. The DNA molecules are the 3.5 kb and 2.2 kb monobiotinylated PCR products used above, and the RNA molecules were synthesized by in vitro transcription from a 4.2 kb long piece of DNA. A short 50-biotinylated RNA oligonucleotide was hybri- dized to one of the ends of both RNA molecules to pro- vide the biotin group for STV binding. The result of our protocol in the absence of STV with the 3.5 kb DNA piece (A) and the single-stranded 4.2 kb RNA (B) molecule is shown in lane 1. The ssRNA migrates approximately at the same speed as 1.5 kb DNA and clearly no dimer for- mation occurs. The result of our protocol in the presence of STV is shown in lane 2: an additional band appears at 5 kb DNA, which is where the ssRNA–DNA dimer is expected. The intensity of this band compared with the others implies an eﬃciency of 40% for the formation of A+B dimers, demonstrating that the eﬃciency of DNA–ssRNA dimers is quite similar to that obtained for the DNA–DNA dimers above. We also demonstrate the use of our protocol in binding a 2.2 kb DNA (A) molecule to a 4.2 kb dsRNA (B) molecule. Lane 4 shows the result after following the protocol without STV: the dsRNA migrates at the same speed at 4.2 kb DNA, and no dimer formation occurs. In lane 3, on the other hand, we observe an additional band at about 6.5 kb, corre- sponding to the heterodimer A+B. From the intensities