Published online 25 July 2008 Nucleic Acids Research, 2008, Vol. 36, No. 16 e104 doi:10.1093/nar/gkn442 End-joining long nucleic acid polymers M. van den Hout, S. Hage, C. Dekker and N. H. Dekker* Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands Received March 17, 2008; Revised June 25, 2008; Accepted June 27, 2008 ABSTRACT Many experiments involving nucleic acids require the hybridization and ligation of multiple DNA or RNA molecules to form a compound molecule. When one of the constituents is single stranded, however, the efficiency of ligation can be very low and requires significant individually tailored optimi- zation. Also, when the molecules involved are very long (>10 kb), the reaction efficiency typically redu- ces dramatically. Here, we present a simple proce- dure to efficiently and specifically end-join two different nucleic acids using the well-known biotin–streptavidin linkage. We introduce a two- step approach, in which we initially bind only one molecule to streptavidin (STV). The second molecule is added only after complete removal of the unbound STV. This primarily forms heterodimers and nearly completely suppresses formation of unwanted homodimers. We demonstrate that the joining efficiency is 50 25% and is insensitive to molecule length (up to at least 20 kb). Furthermore, our method eliminates the requirement for specific complementary overhangs and can therefore be applied to both DNA and RNA. Demonstrated exam- ples of the method include the efficient end-joining of DNA to single-stranded and double-stranded RNA, and the joining of two double-stranded RNA molecules. End-joining of long nucleic acids using this procedure may find applications in bionano- technology and in single-molecule experiments. INTRODUCTION Many experiments involving the manipulation of nucleic acids require the synthesis of complicated molecular con- structs from diﬀerent constituting molecules of DNA or RNA. In single-molecule experiments, for example, con- structs typically contain molecular handles (1–3) or spe- ciﬁc sequences such as DNA hairpins (4,5), DNA–RNA hybrids (6,7), or promoter sequences (8). For certain applications the molecules involved are very long, either because the experimental setup requires a certain mini- mum length (9–11) or because the studied phenomena take place over long distances (12). An example of an experiment requiring very long molecules involves the translocation of single DNA molecules through solid- state nanopores (13–15). Here, molecules are passed through a nanometer-sized hole in a thin membrane by an applied electrical ﬁeld and can be detected by a com- mensurate change in the ionic current through the pore. As this process is typically very fast (13), individual molec- ules have to be very long (>>1 kb) in order to be resolved. Furthermore, increased sophistication of these experi- ments, for instance via integration with optical tweezers (9,16), will permit the interrogation of more complex lengthy molecular constructs. In such cases, the synthesis of molecular constructs can pose a serious challenge to the experimentalist, because standard biochemical reactions can become much less eﬃ- cient for long molecules (17,18). The standard way to construct these molecules is to create complementary overhangs, which hybridize together and can then be ligated to form a stable construct. Although this typically works well for relatively short molecules, the yield of these reactions can drop oﬀ dramatically when the molecules involved reach lengths over 10 kb. Similar techniques using a complementary ‘splint’ molecule to join single- stranded molecules of DNA or RNA have been shown to be useful for joining short oligonucleotides (19,20), but they require the careful selection of the optimal splint molecule and have not demonstrably led to high yields for molecules longer than several hundred bases. To resolve some of these diﬃculties, we here present a straightforward procedure to eﬃciently end-join two arbi- trary molecules in a manner that is independent of the molecule length (up to at least 20 kb). Our method relies on the use of streptavidin (STV) to facilitate eﬃcient end-coupling between two arbitrary nucleic acids A and B, where both A and B can be either DNA or RNA and of arbitrary length. In this scheme the STV acts as a linker between the molecule ends, which can be biotinylated chemically or enzymatically (e.g. using terminal transferase, Klenow polymerase, or poly(A) polymerase) using standard available techniques (21–26). Although a considerable amount of previous work using *To whom correspondence should be addressed. Tel: +31-15-2783219; Fax: +31-15-2781202; Email: n.h.dekker@tudelft.nl 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.