Complementary Overhangs Research Articles

Small Molecule Detection with Ligation-Dependent Light-Up Aptamer Transcriptional Amplification.

ATP and NAD+ are small biomolecules that participate in a variety of physiological functions and are considered as potential biomarkers for disease diagnosis. In this study, we developed a ligation-dependent light-up aptamer transcriptional amplification assay for the sensitive and selective detection of ATP and NAD+. This assay relies on a specific DNA ligase that catalyzes the ligation of a nicked DNA template in the presence of a specific small molecule. We prepared a nicked template consisting of a duplex fragment with an overhang for the T7 promoter region and a single-stranded DNA with a complementary overhang sequence for the Broccoli aptamer. The nicked template was connected using a DNA ligase in the presence of a specific small molecule. The ligation product was subjected to in vitro transcription to amplify the light-up aptamer-mediated fluorescence signals. By integrating the target-dependent ligation and transcription amplification, significant signal amplification was achieved with 5.9 and 142 pM detection limits for ATP and NAD+, respectively. Moreover, good selectivity to discriminate between the target and its analogues was also realized. The application of this method to biological samples was evaluated using human serum and exhibited excellent recovery values.

B-375 Mer-idpcr: A Method for Sequence-specific Measurement of Small RNA Expression and Methylation in Liquid Biopsies

Abstract Background Small RNAs are an important class of biomarkers in liquid biopsy for cancer diagnostics. New discoveries in the field of “epitranscriptomics” have uncovered further functional layers of tumor biology that are ignored by standard gene expression measurement methods. Therefore, there is a pressing need for sequence specific methods that can detect methylation stoichiometry on small RNAs purified from liquid biopsies in a diagnostic setting. Methods To detect small RNAs specifically, we designed structured oligonucleotide adapters that bear reverse complementary overhangs to both the 5′ and 3′ end of target RNAs. After nick-based ligation, reverse transcription under limiting dNTP conditions enables the differential processing of methylated vs unmethylated species. Finally, target specific TaqMan probes are used on a digital PCR based platform for final quantification and calculation of methylation stoichiometry. Results We show that mer-idPCR can detect target RNAs in a sequence-specific manner using synthetic cognate RNA oligos and specificity controls, such as nucleotide variants or non-target miRNAs. Detected RNA is quantified in a linear range with a limit of detection at 40 copies/µL of cDNA. Concurrently, the method can detect 2′-o-methylation in 0%–100% range with a resolution of 10%. mer-idPCR can be applied to RNA extracted from various sources including serum, plasma or whole blood stabilizing tubes. Conclusion mer-idPCR can enable the high resolution, sequence specific quantification of target small RNA species, in addition to the measurement of 2′-o-methylation site status, in small RNAs extracted from peripheral blood. We show the adapter design requirements and discuss optimizations of adapter design and reverse transcription conditions that led to improved specificity, reproducibility, and linearity of this method. mer-idPCR enables the validation and discovery of novel biomarkers for cancer detection in liquid biopsies.

DNA mini-circles reveal effects of DNA topology on CRISPR-Cas9 functions.

CRISPR-Cas9 is an RNA-guided nuclease that has been widely adapted for genome engineering. Prior to Cas9 cleavage, the protospacer of the target DNA must be unwound to form a three-stranded R-loop with a complementary segment of the Cas9-bound guide RNA. R-loop dictates the coordinated conformational changes allosterically controlling Cas9 nuclease activities, thus is the key in target discrimination. Current studies of Cas9 specificity focus primarily on protospacer sequences (i.e., mismatch(es) between the DNA and the RNA guide), while contributions from many collective DNA duplex properties (e.g., flexibility, topological constraints), which could significantly influence DNA unwinding and R-loop structure and dynamics, are not well-understood. In this work, DNA minicircles, which exhibit distinctive features (e.g., ring constraint, curvature), were used to delineate the role of DNA topology on Cas9 function. Two hairpins containing complementary overhangs were ligated to form a single-stranded DNA circle, which was then hybridized with linear complementary strand(s) to generate the double-stranded minicircle (dsMC). With a target imbedded in a 95 base-pair dsMC, the saturating Cas9 cleavage rate, kcat, was found to be ∼1000-fold slower as compared to the corresponding linear construct, although nicking the dsMC resulted in a rate comparable to the linear DNA. Increasing the size of the dsMC to 210 base-pair also led to a significant increase in kcat. The data clearly show that topological constraints imposed by an intact dsMC affect Cas9 cleavage. Work is ongoing to correlate these observed functional effects to the impact of dsMC on the Cas9 R-loop.

Self-Assembled Micro-Sized Hexagons Built from Short DNA in a Crowded Environment.

DNA programmable structures of various morphologies have attracted extensive attention due to their potential for materials science and biomedical applications. Here, we report the formation of micro-sized hexagons via assembly of only one pair of short double-stranded DNA in buffer-salt poly(ethylene glycol) solution. Each DNA strand had complementary bases with a two-base overhang. The procedure of heating and subsequent cooling of blunt-ended double-stranded DNA resulted in different assemblies. These results indicated that end-to-end adhesion at the terminals induced by complementary overhangs were required to construct the hexagonal DNA assemblies. The stable formation of the hexagons was highly dependent on heating temperature. In addition, concentration adjustments of DNA and poly(ethylene glycol) were essential. Circular dichroism spectral measurements and polarization microscopy observations indicated parallel alignment of double-stranded DNA in the hexagonal platelet. Self-assembled micro-sized hexagons composed of simple building blocks may have great potential for future biomedical device development.

Dynamics of Ku and bacterial non-homologous end-joining characterized using single DNA molecule analysis.

We use single-molecule techniques to characterize the dynamics of prokaryotic DNA repair by non-homologous end-joining (NHEJ), a system comprised only of the dimeric Ku and Ligase D (LigD). The Ku homodimer alone forms a ∼2 s synapsis between blunt DNA ends that is increased to ∼18 s upon addition of LigD, in a manner dependent on the C-terminal arms of Ku. The synapsis lifetime increases drastically for 4 nt complementary DNA overhangs, independently of the C-terminal arms of Ku. These observations are in contrast to human Ku, which is unable to bridge either of the two DNA substrates. We also demonstrate that bacterial Ku binds the DNA ends in a cooperative manner for synapsis initiation and remains stably bound at DNA junctions for several hours after ligation is completed, indicating that a system for removal of the proteins is active in vivo. Together these experiments shed light on the dynamics of bacterial NHEJ in DNA end recognition and processing. We speculate on the evolutionary similarities between bacterial and eukaryotic NHEJ and discuss how an increased understanding of bacterial NHEJ can open the door for future antibiotic therapies targeting this mechanism.

The integrin facilitated internalization of fibronectin-functionalized camptothecin-loaded DNA-nanofibers for high-efficiency anticancer effects.

Camptothecin (CMPT) in a free form is extremely cytotoxic as well as hydrophobic drug, and is considered to be highly contagious for systemic administration. The fibronectin (FN)-functionalized DNA-based nanocarrier has been designed to load CMPT and target integrin (αvβ3) receptors which are highly expressed on the A549 cancer cells. Here, we report DNA nanocarrier in the form of DNA-nanofibers (DNA-NFs) capable of loading CMPT via strand intercalation in the GC (base pairs)-rich regions of the DNA duplex. Hence, our keen purpose was to explore the potential of DNA-NFs to load CMPT and assess the improvements of the outcomes in terms of enhanced therapeutic effects to integrin-rich A549 cancer cells with reduced cytotoxic effects to integrin-lacking HEK293 cells. DNA-NFs were formulated as a polymer of DNA triangles. DNA triangles arranged in a programmed way through the complementary overhangs present at the vertices. DNA triangles were primarily obtained through the annealing of the freshly circularized scaffold strands with the three distinct staple strands of specific sequences. The polymerized triangular tiles instead of forming two-dimensional nanosheets underwent self-coiling to give rise to DNA-NF-shaped structures. Flow cytometry and MTT assays were performed to observe cytotoxic and apoptotic effects on integrin-rich A549 cancer cells compared with the integrin-deficient HEK293 cells. AFM, native-page, and confocal experiments confirmed the polymerization of DNA triangles and the morphology of the resulting nanostructures. AFM and confocal images revealed the length of DNA-NFs to be 3-6μm and the width from 70 to 110nm. CMPT loading (via strands intercalation) in GC-rich regions of DNA-NFs and the FN functionalization (TAMRA tagged; red fluorescence) via amide chemistry using amino-modified strands of DNA-NFs were confirmed through the UV-shift analysis (> 10nm shift) and confocal imaging. Blank DNA-NFs were found to be highly biocompatible in 2-640μM concentrations. MTT assay and flow cytometry experiments revealed that CMPT-loaded DNA-NFs showed a dose-dependent decrease in the cell viability to integrin-rich A549 cancer cells compared with the integrin-deficient HEK293 cells. Conclusively, FN-functionalized, CMPT-loaded DNA-NFs effectively destroyed integrin-rich A549 cancer cells in a targeted manner compared with integrin-deficient HEK293 cells. Grapical abstract.

Classifying Cell Types with DNA-Encoded Ligand-Receptor Interactions on the Cell Membrane.

Clustering, endocytosis, and intracellular transport of molecules on the cell membrane are critically dependent on the type of cells. However, the membrane-associated redistribution of molecules has not been exploited to realize cell classification for diagnostic purposes. Here, we develop a set of DNA-encoded artificial receptors and ligands to monitor the cell membrane redistribution. In this system, a cholesterol-modified single-stranded DNA strand serves as the receptor localized on the membrane, and a tetrahedral DNA framework (TDF) nanostructure with a complementary overhang serves as the ligand. The DNA-encoded receptor-ligand interaction is highly orthogonal, mimicking the dynamics of natural receptors and ligands on cells. We demonstrate that the dynamics of membrane redistribution can be resolved by the dual-color fluorescent patterns of the receptor-ligand interactions in a single image, which can be exploited to classify cell lines with high fidelity. This DNA-encoded method thus holds great promise for cell typing and diagnosis.

In Vitro Construction of Large-scale DNA Libraries from Fragments Containing Random Regions using Deoxyinosine-containing Oligonucleotides and Endonuclease V.

Efficient and precise construction of DNA libraries is a fundamental starting point for directed evolution of polypeptides. Recently, several in vitro selection methods have been reported that do not rely on cells for protein expression, where peptide libraries in the order of 1013 species are used for in vitro affinity selection. To maximize their potential, simple yet versatile construction of DNA libraries from several fragments containing random regions without bacterial transformation is essential. To address this issue, we herein propose a novel DNA construction methodology based on the use of polymerase chain reaction (PCR) primers containing a single deoxyinosine (I) residue near their 5' end. Treatment of the PCR products with endonuclease V generates 3' overhangs with customized lengths and sequences, which can be ligated accurately and efficiently with other fragments having exactly complementary overhangs. As a proof of concept, we constructed an artificial gene library of single-domain antibodies from four DNA fragments.

Thermus thermophilus DNA Ligase Connects Two Fragments Having Exceptionally Short Complementary Termini at High Temperatures.

Thermus thermophilus DNA ligase (Tth DNA ligase) is widely employed for cloning, enzymatic synthesis, and molecular diagnostics at high temperatures (e.g., 65 °C). It has been long believed that the complementary ends must be very long (e.g., >30 bp) to place two DNA fragments nearby for the ligation. In the current study, the length of the complementary portion was systematically varied, and the ligation efficiency was evaluated using the high resolution melting (HRM) method. Unexpectedly, very short oligonucleotides (7-10 nt) were successfully ligated on the complementary overhang attached to a dsDNA at 70 °C. Furthermore, sticky ends with the overhang of only 4 nt long, available after scission with many restriction enzymes, were also efficiently ligated at 45-70 °C. The ligation yield for the 6-nt-long sticky ends was as high as 80%. It was concluded that Tth DNA ligase can be used as a unique tool for DNA manipulation that cannot be otherwise easily accomplished.

A DNA Origami Platform for Single-Pair Förster Resonance Energy Transfer Investigation of DNA-DNA Interactions and Ligation.

DNA double-strand breaks (DSBs) pose an everyday threat to the conservation of genetic information and therefore life itself. Several pathways have evolved to repair these cytotoxic lesions by rejoining broken ends, among them the nonhomologous end-joining mechanism that utilizes a DNA ligase. Here, we use a custom-designed DNA origami nanostructure as a model system to specifically mimic a DNA DSB, enabling us to study the end-joining of two fluorescently labeled DNA with the T4 DNA ligase on the single-molecule level. The ligation reaction is monitored by Förster resonance energy transfer (FRET) experiments both in solution and on surface-anchored origamis. Due to the modularity of DNA nanotechnology, DNA double strands with different complementary overhang lengths can be studied using the same DNA origami design. We show that the T4 DNA ligase repairs sticky ends more efficiently than blunt ends and that the ligation efficiency is influenced by both DNA sequence and the incubation conditions. Before ligation, dynamic fluctuations of the FRET signal are observed due to transient binding of the sticky overhangs. After ligation, the FRET signal becomes static. Thus, we can directly monitor the ligation reaction through the transition from dynamic to static FRET signals. Finally, we revert the ligation process using a restriction enzyme digestion and religate the resulting blunt ends. The here-presented DNA origami platform is thus suited to study complex multistep reactions occurring over several cycles of enzymatic treatment.

Fabricating and Actuating DNA Origami Mechanisms

DNA origami enables the precise fabrication of nanoscale geometries. We demonstrate an approach to engineer complex and reversible motion of nanoscale DNA origami mechanisms. Following a traditional macroscopic machine design approach, we developed flexible DNA origami rotational and linear joints that integrate stiff double-stranded DNA components and flexible single-stranded DNA components to constrain motion along a single degree of freedom and demonstrate the ability to tune flexibility and range of motion. Linear motion was achieved by folding a tube concentrically around a track through a novel approach of programmed sequential folding so the track folds first, and then the tube is constrained to assemble around the track. Multiple joints were then integrated into various higher order mechanisms including a crank–slider that couples rotational and linear motion. We have also demonstrated multiple actuation methods to achieve reversible conformational changes via input strands to form new connections distributed throughout the mechanisms. One approach uses stable input strand connections with DNA strand displacement, and we have established an approach for reversible actuation that relies on transient binding of many weak affinity input strands. In this case, the reverse actuation can be achieved simply by removing the input strands from solution. Also, we have recently developed a similar actuation method using weak complementary overhang connections that can that can be controlled by adjusting cation concentration. We expect these approaches can improve actuation times because they do not rely on strand displacement. In addition, with both approaches, we can tune the dynamics to see conformational changes in specific ranges of concentrations. Our results demonstrate programmable motion of 2D and 3D DNA origami mechanisms constructed following a macroscopic machine design approach with steps towards fast mechanical actuation.

Melting transition in lipid vesicles functionalised by mobile DNA linkers.

We study phase behaviour of lipid-bilayer vesicles functionalised by ligand-receptor complexes made of synthetic DNA by introducing a modelling framework and a dedicated experimental platform. In particular, we perform Monte Carlo simulations that combine a coarse grained description of the lipid bilayer with state of art analytical models for multivalent ligand-receptor interactions. Using density of state calculations, we derive the partition function in pairs of vesicles and compute the number of ligand-receptor bonds as a function of temperature. Numerical results are compared to microscopy and fluorimetry experiments on large unilamellar vesicles decorated by DNA linkers carrying complementary overhangs. We find that vesicle aggregation is suppressed when the total number of linkers falls below a threshold value. Within the model proposed here, this is due to the higher configurational costs required to form inter-vesicle bridges as compared to intra-vesicle loops, which are in turn related to membrane deformability. Our findings and our numerical/experimental methodologies are applicable to the rational design of liposomes used as functional materials and drug delivery applications, as well as to study inter-membrane interactions in living systems, such as cell adhesion.

Uncovering the thermodynamics of monomer binding for RNA replication.

The nonenzymatic replication of primordial RNA is thought to have been a critical step in the origin of life. However, despite decades of effort, the poor rate and fidelity of model template copying reactions have thus far prevented an experimental demonstration of nonenzymatic RNA replication. The overall rate and fidelity of template copying depend, in part, on the affinity of free ribonucleotides to the RNA primer–template complex. We have now used 1H NMR spectroscopy to directly measure the thermodynamic association constants, Kas, of the standard ribonucleotide monophosphates (rNMPs) to native RNA primer–template complexes. The binding affinities of rNMPs to duplexes with a complementary single-nucleotide overhang follow the order C > G > A > U. Notably, these monomers bind more strongly to RNA primer–template complexes than to the analogous DNA complexes. The relative binding affinities of the rNMPs for complementary RNA primer–template complexes are in good quantitative agreement with the predictions of a nearest-neighbor analysis. With respect to G:U wobble base-pairing, we find that the binding of rGMP to a primer–template complex with a 5′-U overhang is approximately 10-fold weaker than to the complementary 5′-C overhang. We also find that the binding of rGMP is only about 2-fold weaker than the binding of rAMP to 5′-U, consistent with the poor fidelity observed in the nonenzymatic copying of U residues in RNA templates. The accurate Ka measurements for ribonucleotides obtained in this study will be useful for designing higher fidelity, more effective RNA replication systems.

Single Molecule Observation of Cyclization of Short DNA Duplex

In the presented work, a single molecule DNA cyclization assay was used to follow the looping kinetics of single DNA 83 bp molecules, utilizing single molecule fluorescence energy transfer (smFRET) technique. The assay was first prepared in a Na+ free condition and the majority of the DNA was in its unlooped form. A sudden Na+ jump was introduced at different concentrations (0.05-1.75M) and finally yielded DNA in its looping state by annealing the complementary single-strand overhangs of the assay. Looping and unlooping rates were obtained from the kinetic measurements. The result shows a positive and negative linear dependence of the Na+ concentration to the looping and unlooping rate, respectively, until they reach a plateau at 500 mM. The plateau persists until about 1M. For concentrations beyond 1M, an immoderate increase in looping rate is noticed while the unlooping rate does so gradually. Above 1M Na+ there is a preference of looping events that is attributed to the increase of the annealing rate of the overhangs rather than increased flexibility, consistent with earlier studies by Ibrahim Cisse et al. (2012).A protein mediated cyclization assay was also used in experiments with HU protein in which a dramatic increase in the looping rate is noticeable. However in high HU concentration, looping is prohibited implying filament formation.

A Practical Comparison of Ligation-Independent Cloning Techniques

The precise assembly of specific DNA sequences is a critical technique in molecular biology. Traditional cloning techniques use restriction enzymes and ligation of DNA in vitro, which can be hampered by a lack of appropriate restriction-sites and inefficient enzymatic steps. A number of ligation-independent cloning techniques have been developed, including polymerase incomplete primer extension (PIPE) cloning, sequence and ligation-independent cloning (SLIC), and overlap extension cloning (OEC). These strategies rely on the generation of complementary overhangs by DNA polymerase, without requiring specific restriction sites or ligation, and achieve high efficiencies in a fraction of the time at low cost. Here, we outline and optimise these techniques and identify important factors to guide cloning project design, including avoiding PCR artefacts such as primer-dimers and vector plasmid background. Experiments made use of a common reporter vector and a set of modular primers to clone DNA fragments of increasing size. Overall, PIPE achieved cloning efficiencies of ∼95% with few manipulations, whereas SLIC provided a much higher number of transformants, but required additional steps. Our data suggest that for small inserts (<1.5 kb), OEC is a good option, requiring only two new primers, but performs poorly for larger inserts. These ligation-independent cloning approaches constitute an essential part of the researcher's molecular-tool kit.

Impact of siRNA Overhangs for Dendrimer-Mediated siRNA Delivery and Gene Silencing

Small interfering RNA (siRNA) have attracted considerable attention, as compelling therapeutics providing safe and competent delivery systems are available. Dendrimers are emerging as appealing siRNA delivery vectors thanks to their unique, well-defined architecture and the resulting cooperativity and multivalency confined within a nanostructure. We have recently disclosed the structurally flexible fifth-generation TEA-core PAMAM dendrimer (G5) as an effective nanocarrier for delivery of sticky siRNA bearing long complementary sequence overhangs (dA)n/(dT)n (n = 5 or 7). Here, using combined experimental/computational approaches, we successfully clarified (i) the underlying mechanisms of interaction between the dendrimer nanovector G5 and siRNA molecules bearing either complementary or noncomplementary sequence overhangs of different length and chemistry and (ii) the impact of siRNA overhangs contributing toward the improved delivery potency. Using siRNA with complementary overhangs offer the best action in term of gene silencing through the formation of concatemers, that is, supramolecular structures resulting from synergistic and cooperative binding via (dA)n/(dT)n bridges (n = 5 or 7). On the other hand, although siRNA bearing long, noncomplementary overhangs (dA)n/(dA)n or (dT)n/(dT)n (n = 5 or 7) are endowed with considerably higher gene silencing potency than normal siRNA with (dT)2/(dT)2, they remain less effective than their sticky siRNA counterparts. The observed gene silencing potency depends on length, nature, and flexibility of the overhangs, which behave as a sort of clamps that hold and interact with the dendrimer nanovectors, thus impacting siRNA delivery performance and, ultimately, gene silencing. Our findings can be instrumental in designing siRNA entities with enhanced capability to achieve effective RNA interference for therapeutic applications.

A modified plasmid vector pCMV–3Tag–LIC for rapid, reliable, ligation-independent cloning of polymerase chain reaction products

Here we present a modified vector pCMV–3Taq–LIC for a rapid, simple, and relatively cheap method to build expression constructs. After being digested by Nt.BspQI and EcoRV, a lineal vector with specific 11-base single overhangs is obtained. Polymerase chain reaction (PCR) products with complementary overhangs are created by building appropriate extensions into the primers and treating them with T4 DNA polymerase. The annealing of the insert and the vector is performed in the absence of ligase by simple mixing of the DNA fragments. This process is very specific because only the desired products can form. Using this vector, we successfully constructed hnRNP K full-length complementary DNA (cDNA) expression plasmid.

DNA barcode profiling: a new platform for the investigation of genome integrity

The term 'DNA barcode' has been used in various contexts. Here we use it to describe short (8-14 bases) single stranded DNA molecules of a fixed length (either 8, 9 bases long) that were isolated in various ways from a biological sample with the purpose of making a digital profile of expressed mRNAs, miRNAs, 16S RNAs, for example, or other information-relevant nucleic acids in a biological sample. Thus, no a priori assumptions or prior knowledge is required when using the DNA barcode technology. A single stranded DNA barcode can be detected and quantified by ligation from both ends simultaneously on a digital microarray, it can be detected as one unit in a single nanopore event, or it can be sequenced on a genome sequencer. Every detection/quantification method has pros and cons in terms of cost and efficiency. Genomic Expression Inc has developed its first universal digital microarray-based product for detection and quantification of DNA barcodes. Detection on the universal digital microarray is based upon ligation of the DNA barcodes to complementary overhang sequences of partly double stranded probes at one end of the DNA barcodes and ligation to the complementary overhang of another probe in solution that is coupled to a fluorochrome at the other end of the DNA barcode. Specialized software is designed to enhance the extraction of information from a profile of DNA barcodes and translate this information to the original sample depending upon how the DNA barcodes were extracted from the sample. The poster will provide a few examples of how a profile of DNA barcodes can be obtained from a biological sample and also show some data from a 'SAGE- on-a-chip' experiment. These data show that the DNA barcode technology using a universal digital array has the potential of being significantly more sensitive than a conventional SAGE profile. A lot of expressed DNA barcode tags found at relatively high abundance using the DNA barcode technology are not picked up by a SAGE profile. The DNA barcode technology offers a new type of cost-effective open-ended solution for such diverse applications as genome-wide methylation profiling, environmental microbiome profiling, expression profiling and single gene mutation detection, with only minor adjustments in how the DNA barcodes are obtained from a biological sample.

Requirement for XLF/Cernunnos in alignment-based gap filling by DNA polymerases λ and μ for nonhomologous end joining in human whole-cell extracts

XLF/Cernunnos is a core protein of the nonhomologous end-joining pathway of DNA double-strand break repair. To better define the role of Cernunnos in end joining, whole-cell extracts were prepared from Cernunnos-deficient human cells. These extracts effected little joining of DNA ends with cohesive 5′ or 3′ overhangs, and no joining at all of partially complementary 3′ overhangs that required gap filling prior to ligation. Assays in which gap-filled but unligated intermediates were trapped using dideoxynucleotides revealed that there was no gap filling on aligned DSB ends in the Cernunnos-deficient extracts. Recombinant Cernunnos protein restored gap filling and end joining of partially complementary overhangs, and stimulated joining of cohesive ends more than twentyfold. XLF-dependent gap filling was nearly eliminated by immunodepletion of DNA polymerase λ, but was restored by addition of either polymerase λ or polymerase μ. Thus, Cernunnos is essential for gap filling by either polymerase during nonhomologous end joining, suggesting that it plays a major role in aligning the two DNA ends in the repair complex.

End-joining long nucleic acid polymers

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 optimization. Also, when the molecules involved are very long (>10 kb), the reaction efficiency typically reduces dramatically. Here, we present a simple procedure 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 examples 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 bionanotechnology and in single-molecule experiments.