Identification of Extrachromosomal Linear microDNAs Interacted with microRNAs in the Cell Nuclei

Extrachromosomal DNA exists in two forms: Covalently closed circular and linear. While diverse types of circular extrachromosomal DNA have been identified with validated in vivo functions, little is known about linear extrachromosomal DNA. In this study, we identified small, single-stranded linear extrachromosomal DNAs (SSLmicroDNAs) in the nuclei of mouse hearts, mouse brains, HEK293, and HeLa cells. We used a pull-down system based on the single-stranded DNA binding protein RecAf. We found that SSLmicroDNAs aligned predominantly to intergenic and intragenic regions of the genome, owned a variety of single nucleotide polymorphism sites, and strongly associated with H3K27Ac marks. The regions were tens to hundreds of nucleotides long, periodically separated by AT, TT, or AA dinucleotides. It has been demonstrated that SSLmicroDNAs in the nuclei of normal cells target microRNAs, which regulate biological processes. In summary, our present work identified a new form of extrachromosomal DNAs, which function inside nuclei and interact with microRNAs. This finding provides a possible research field into the function of extrachromosomal DNA.


Introduction
Extrachromosomal DNA-DNA molecules separated from chromosomes-exist in two main forms: Covalently closed circular and linear DNA [1][2][3][4][5]. Mitochondrial DNA are located in mitochondria and represent typical extrachromosomal circular DNA (eccDNA). This type of DNA encodes functional genes and contributes to the nuclear genomes' instability [6]. Small polydispersed circular DNA, double minute chromosomes, episomes, minichromosomes, autonomously replicating sequences, telomeric-circles, B-and T-cell receptor excision circles, and extrachromosomal elements induced by c-myc oncogene deregulation and resulting in genomic instability were discovered as extrachromosomal circular DNA molecules with unique properties and relevant in vivo functions, which are produced randomly or non-randomly [1,[7][8][9][10]. In recent years, an increasing number of eccDNAs have been identified in many organisms. Preliminary research links eccDNAs to cancer [11,12]. Recently, much attention has focused on a class of small circular extrachromosomal DNAs (microDNAs), with short length and unique features. microDNAs have been found ubiquitously expressed in mammalian cells, including mouse brain, heart, kidney, liver, lung, skeletal muscle, sperm,

SSLmicroDNA Library Construction and Sequencing
Total single-stranded linear DNAs were ligated with two specific double-stranded adaptors-adaptor A or adaptor B. The adaptor A forward sequence was: 5 -CACACTCTTTCCCTACACGACGCTCTTCCGATCTTTGCNNNNNN-3 , and the reverse sequence was 5 -GCAAAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTG-3 . The adaptor B forward sequence was 5 -NNNNNNGTTCAGAGTTCTGCGACAGGAGAGGTCGTATGCCGTCTTCTGCTTG-3 , and the reverse sequence was 5 -CAAGCAGAAGACGGCATACGACCTCTCCTGTCGCAGAACTCTGAAC-3 . The adaptors could only ligate to single-stranded linear DNA through sticky ends and six random  The complementary strand was synthesized and amplified by a PCR  reaction with the forward primer 5 -CAAGCAGAAGACGGCATACGA-3 and the reverse primer  5 -ACACTCTTTCCCTACACGAC-3 . DNA mixtures ranging from less than 500 bp, 500-1000 bp,  and 1000-2000 bp were collected and cloned into the pZeroback T vector (TianGen biotech co., LTD., Beijing, China). Sequencing was performed using the TSINGKE Biological Technology.

Atomic Force Microscopy
DNA was imaged using atomic force microscopy, the experiment was conducted following the protocol described in Reference [28]. Briefly, a drop of DNA (5 ng/µL) with 5 mM MgCl 2 was placed on the surface of freshly cleaved mica, and left for 2 min at room temperature. The mica was then rinsed with 1 mL water, blotted with filter paper, and dried by the flow of compressed nitrogen for 2 min. Samples in mica were scanned using a Digital Instrument's MultiMode scanning probe on a Nanoscope IIIa (Veeco, New York, NY, USA) microscope operating in Tapping Mode.

Fluorescence in situ Hybridization
Fluorescence in situ hybridization was performed following after modifying the method described in Reference [29]. Briefly, cells were grown on glass coverslips, coated with poly-L-lysine (Sigma-Aldrich, St. Louise, MO, USA), and fixed with 4% paraformaldehyde for 30 min at room temperature. They were treated consecutively with 4.5% sucrose buffer for 1 h four times, fresh 0.1 M phosphate buffered saline (PBS) for 5 min, 0.3% Triton X-100 for 5 min, 0.1 M PBS for 5 min two times, and 2 µg/mL proteinase K (diluted by 50 mM Tris and 2 mM calcium chloride) at 37 • C for 20 min. The cells were then fixed again, and treated with 0.25% acetic anhydride and 0.1 M ethanolamine for 10 min, 50% formamide/2*SSC (0.3MNaCl, 30mM sodium citrate) for 10 min at room temperature, and then incubated at 37 • C for 15 min. Pre-hybridization was performed using 100 µg/mL salmon sperm DNA and incubated at 37 • C for 2 h. Finally, the samples were incubated with 400 ng/mL of probe, in hybridization buffer, at 37 • C overnight. The 293mid-28 probe was modified using 6-FAM at the 5 end, and its sequence was 5 -AGATCGCACTACTGCACTCCAGCCTGGGTGACAGAGTAAGACTTCGTCTC-3 . The HeLamid-18 probe was modified using 6-TAMRA at the 5 end and its sequence was 5 -GATGGGGGTAAATATCCAGGCTTTCCGCTCTGCTTCCATTGACACCCAAT-3 .

Cell Culture and Isolation of Nuclei
HEK293 and HeLa cells were grown in Dulbecco's modified Eagle's medium (DMEM; Invitrogen TM life technology) supplemented with 10% Fetal bovine serum (FBS), 100 units/mL penicillin, and 100 µg/mL streptomycin and incubated at 37 • C and 5% CO 2 . Nuclei were isolated according to a previously described method with minor modifications [7]. In this study, cells were isolated from mouse hearts (adult males, 8-12 weeks of age), mouse brains (adult males, 8-12 weeks of age), HEK293 cells, or HeLa cells. Nuclei were extracted from tissue or cell lines using sucrose gradient ultracentrifugation. Fresh tissue or cell lines were homogenized using a wheaton douncer, in buffer A (20 mM hepes pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EGTA, 1 mM EDTA, 1 mM DTT, 0.1 mM PMSF, and 250 mM sucrose). Cell debris was removed and intact cells were pelleted after centrifugation at 800 rpm for 10 min. Nuclei were precipitated through centrifugation at 3000 rpm for 15 min.

Sequence Analysis
Genome mapping, H3K27Ac activity, single nucleotide polymorphism (SNP) site, and conservation level analysis were performed in the UCSC (University of California Santa Cruz) genome browser. The length distribution and GC content were analyzed by Mega software. Base-pairing analysis was performed using the bioinformatics program RNAhybrid. All statistical analyses were performed with the SPSS 16.0 software.

Detection of the Endogenous Linear microDNAs
HEK293 cells were transfected with biotinylated 293mid-62 antisense sequence 5 -TTGAGCATTTACCATGTTCTGGGCAGTGTGGAAGGCCTTGGGTGTATAATGTGAGCATGTG CCTGACCTGTGCTGATGGAGGAGACAAATA-3 , which was 20 nt shorter at the 5 -end than 293mid-62. The pull-down assay, based on interactions between biotin and streptavidin (described in the method-pull-down assay and quantitative real-time PCR), was performed to purify 293mid-62. The complementary strand was synthesized and amplified by a PCR reaction with the forward primer 5 -CTTTGATCAGCATTATTTATTTTG-3 and the reverse primer 5 -TATTTGTCTCCTCCATCAGCACAGG-3 .
The amplified product was cloned into the pLB Vector (TIANGEN biotech) and sequenced.

Transfection and Detection of Exogenous SSLmicroDNAs and Reverse Strands
The 293mid-28, HeLamid-18, and their reverse strands were synthesized by the Sangon Biotech company. The 293mid-28 and its reverse strands were modified using 6-FAM at the 5 end. HeLamid-18 and its reverse strands were modified using 6-TAMRA at the 5 end. The sequence of 293mid-28 was 5 -TTTTTTTTTTTTTTTGAGACGAAGTCTTACTCTGTCACCCAGGCTGGAGTGCAGTAGTGCGA TCTCAGCTCACTGCAACCTCCGCCTCCCAG-3 , and its reverse strand sequence was 5 -CTGGGAGGCGGAGGTTGCAGTGAGCTGAGATCGCACTACTGCACTCCAGCCTGGGTGACA GAGTAAGACTTCGTCTCAAAAAAAAAAAAAAA-3 .
The sequence of HeLamid-18 was 5 -CACCCAGCAGCAACAGGGGTCCCCCACCCCATTGGGTGTCAATGGAAGCAGAGCGGAAAG CCTGGATATTTACCCCCATCTAGAAGTAACAAG-3 , and its reverse strand sequence was 5 -CTTGTTACTTCTAGATGGGGGTAAATATCCAGGCTTTCCGCTCTGCTTCCATTGACACCCAAT GGGGTGGGGGACCCCTGTTGCTGCTGGGTG-3 . HEK293 and HeLa cells were grown on glass coverslips coated with poly-L-lysine (Sigma-Aldrich). The transfection was performed using the transfection reagent Fugene (Promega, Fitchburg, WI, USA), according to the manufacturer's instructions. Cells were fixed with cold methanol at −20 • C for 5-10 min, and stained with DAPI. They were analyzed using a laser confocal fluorescence microscope.

Small, Single-Stranded Linear Extrachromosomal DNAs (named SSLmicroDNAs) were Identified in Cell Nucleus
We purified single-stranded linear DNA using the single-stranded DNA binding protein RecAf. The DNA was extracted from the nuclei of adult mouse hearts, brains, HEK293 cells, and HeLa cells [30,31] (Figure 1a). The purified single-stranded linear DNAs were further identified using atomic force microscopy (AFM; Figure 2a). Smear-like band distributions with a broad range were observed in agarose gel electrophoresis analysis (Figure 2b). coverslips coated with poly-L-lysine (Sigma-Aldrich). The transfection was performed using the transfection reagent Fugene (Promega, Fitchburg, WI, USA), according to the manufacturer's instructions. Cells were fixed with cold methanol at −20 °C for 5-10 min, and stained with DAPI. They were analyzed using a laser confocal fluorescence microscope.

Small, Single-Stranded Linear Extrachromosomal DNAs (named SSLmicroDNAs) were Identified in Cell Nucleus.
We purified single-stranded linear DNA using the single-stranded DNA binding protein RecAf. The DNA was extracted from the nuclei of adult mouse hearts, brains, HEK293 cells, and HeLa cells [30,31] (Figure 1a). The purified single-stranded linear DNAs were further identified using atomic force microscopy (AFM; Figure 2a). Smear-like band distributions with a broad range were observed in agarose gel electrophoresis analysis (Figure 2b). The single-stranded linear DNAs were pulled down by the RecAf protein. RNAs and single-stranded linear (SSL) DNAs were isolated through pulldown system based on RecAf protein. microRNA array analysis was performed with the RNA samples. Following ligation with two adaptors, the singlestranded linear DNAs libraries were constructed and sequenced. (b) The composition of adaptor A and adaptor B. Adaptors composed of end markers and random bases. N represents a random base. TTTGC and GCAAA were end markers in adaptor A or B. Ni-NTA.   data set 4). These data showed that mouse heart-derived linear DNA had a 16-579 nt length distribution with a peak at approximately 130 nt (Figure 2d); 50-400 nt length for mouse brain-derived linear DNA (Figure 2e); and 20-290 nt length for those derived from HEK293 cells, of which >90% were distributed between 40-150 nt (Figure 2f). However, compared to normal tissue, the length distribution of linear DNAs from HeLa cells was irregular, without a defined length peak (Figure 2d-g). Since linear DNAs have molecular weights ranging from tens to thousands, with peaks at the hundreds mark, we named them single-stranded linear microDNAs (SSLmicroDNAs). SSLmicroDNAs derived from mouse hearts, mouse brains, HEK293 cells, and HeLa cells were named MHSSLmid, MBSSLmid, 293SSLmid, and HeLaSSLmid, respectively.

SSLmicroDNAs Exhibited a Series of Unique Features and Their Origins Hypothesis was Put Forward
To understand the nature of SSLmicroDNAs, we performed detailed characterization using statistical and bioinformatic methods. We conducted a comparative analysis of all 383 SSLmicroDNA sequences in the UCSC genome browser ( Figure S3). The results showed >90% of these sequences were located in non-coding regions, including intergenic and intragenic regions (Figure 3a). Analysis of microDNA loci and their neighboring regions showed that the 42.5% GC content of HeLaSSLmids was slightly higher than the 37.5% GC content of their 500 bp up or downstream neighboring regions (Figure 3b). MHSSLmids had a similar GC content to HeLaSSLmids, while MBSSLmids and 293SSLmids did not (Figure 3b). Remarkably, AT, TT, or AA dinucleotides appeared periodically, every 10 bp along the microDNA sequence, and were identified in approximately 76.64% of 293SSLmids, 64.84% of MHSSLmids, 57.01% of MBSSLmids, and 38.57% of HeLaSSLmids (Figure 3c). The periodicity of microDNAs isolated from normal tissues was much greater than those from tumor cells (Figure 3c). A previous study revealed that AT, TT, or AA dinucleotide periodicity usually appeared in sequences assembled into nucleosomes [32]. Therefore, we hypothesized that SSLmicroDNAs may originate from nucleosomes.
Single nucleotide polymorphisms (SNPs) refer to changes in single nucleotides of DNA, which lead to genome diversity and instability [33]. Our results showed that~50% of microDNAs in mouse heart, mouse brain, and HEK293 cells, and >90% in HeLa cells were located in SNP-rich regions-2 kb upstream or downstream (Figure 3d,f). H3K27Ac chromatin marks, usually appear at the site of active regulatory elements, such as gene enhancers and promoters [34]. Approximately 30% of SSLmicroDNAs from HEK293 and HeLa cells were located near high-H3K27Ac-activity regions (Figure 3e,f), which led us to hypothesize that H3K27Ac-marked regions could be a source for SSLmicroDNA production. Taken together, these data indicate that SSLmicroDNAs are likely generated from regions with strong H3K27Ac marks, and frequent single nucleotide polymorphisms.
To further confirm the type of purified extrachromosomal DNA; single-stranded or linear, we designed a pull-down system based on the interaction between biotin and streptavidin (Supplementary Figure S1). The results showed that 293mid-62 was pulled down by a 293mid-62 probe modified with a biotin tag at the 5 -end, and HeLamid-1 was pulled down by its respective probe (Figure 4a).   generated from regions with strong H3K27Ac marks, and frequent single nucleotide polymorphisms. To further confirm the type of purified extrachromosomal DNA; single-stranded or linear, we designed a pull-down system based on the interaction between biotin and streptavidin (Supplementary Figure S1). The results showed that 293mid-62 was pulled down by a 293mid-62 probe modified with a biotin tag at the 5'-end, and HeLamid-1 was pulled down by its respective probe (Figure 4a).  The 293mid-62 probe with a biotin tag at the 5 -end was transfected into HEK293 cells, and HeLamid-1 probe with a biotin tag at the 5 -end was transfected into HeLa cells. Then the pulldown system based on interaction between biotin and streptavidin was performed and the 293mid-62 and HeLamid-1 were detected by sequencing. (b,c) Detection of SSLmicroDNA subcellular localization. HEK293 cells were transfected with 293mid-28 or its reverse strands labeled with a FAM at the 5 -end (b), and HeLa cells were transfected with HeLamid-18 or its reverse strands labeled with a TAMRA at the 5 -end (c). The nuclei were stained by DAPI (the dark blue area). The light blue dots represented 293mid-28 or its reverse strands. The red dots represented HeLamid-18 or its reverse strands. The Bar = 10 µm. (d) Fluorescence in situ hybridization (FISH) analysis of SSLmicroDNA localization in cell nucleus. 293mid-28 was detected by its probes labeled with FAM at the 5 -end in HEK293 cell nucleus. HeLamid-18 was detected by its probes labeled with TAMRA at the 5 -end in HeLa cell nuclei. The nuclei were stained by DAPI (the dark blue area). The light blue dots represented 293mid-28. The red dots represented HeLamid-18. Bar = 20 µm. (e) Analysis of the conservation level of SSLmicroDNA sequences. Species number represents the number of species in which SSLmicroDNAs were conserved. f, Summary of the conservation level of SSLmicroDNAs. The SSLmicroDNAs that were conserved in more than one species were counted.

Cell Nuclei-Located SSLmicroDNAs Owned a High Conservation Level
We then studied the subcellular localization of microDNAs, 293mid-28 and HeLamid-18 were chosen randomly. Twenty-four hours post transfection, both 293mid-28 and its reverse strand were observed in the nuclei of HEK293 cells (Figure 4b). HeLamid-18 and its reverse strand were transfected into HeLa cells, and were also found in the nucleus (Figure 4c). These results were further confirmed by fluorescence in situ hybridization (FISH), showing endogenous 293mid-28 and HeLamid-18 in the nuclei (Figure 4d).
After analyzing the conservation levels of SSLmicroDNAs, we found that >80% of HEK293SSLmicroDNAs,~60% of MHSSLmicroDNAs, and~40% of MBSSLmicroDNAs were conserved between two or more species, whereas only 32.86% of HeLaSSLmicroDNAs shared this feature (Figure 4e,f). These results indicated that SSLmicroDNAs in normal tissues or cells are slightly more conserved compared to their tumor-derived counterparts.

SSLmicroDNAs Interacted with microRNAs in the Cell Nucleus
Many miRNAs are distributed in nuclei, where they can regulate chromatin remodeling, transcriptional silencing, mRNA alternative splicing, and microRNA maturation [23][24][25]27]. The abundance of SSLmicroDNAs in the nuclei of normal tissues and cells suggested a potential participation in microRNA regulatory pathways. Therefore, we studied the interaction between microDNAs and microRNAs. MicroRNAs were extracted from the nuclear pull-down product via the single-stranded DNA binding protein RecAf and analyzed using microRNA arrays. MicroRNA arrays identified 421 mouse heart (Table S1), 285 mouse brain (Table S2), and 1101 HEK293 microRNAs (Table S3). We analyzed all of the microRNA and SSLmicroDNA sequences using the bioinformatics program RNAhybrid. The results showed that microDNAs pair well to at least one microRNA (Figure 5a-c, and Supplementary Figures S4-S6). Among all the microDNA-microRNA sequence alignments, we detected~45.76% hybridization with more than 20 kcal/mol free energy in the mouse brain library, and 36.38% in the mouse heart library (Figure 5d). Furthermore, 24.41% of the hybridizations between microDNAs and microRNAs in mouse hearts, 28.97% in mouse brains, and 27.27% in HEK293 cells exhibited more than 6 bp-continuous base pairing at microRNA seed regions. At any given location, 10 bp-continuous base pairing appeared in 56.69% of the hybridizations in the mouse heart, 58.88% in mouse brains, and 66.23% in HEK293 cells (Figure 5e). The microRNAs seed region is a critical recognition and functional site for targeting to mRNAs [35]. Therefore, we hypothesized that this was also a site for microDNAs interaction with microRNAs in the cell's nucleus. To validate this hypothesis, we performed pull-down experiments, and showed that mir-1273g-3p was pulled down by the 293mid-28, and mir-5096 was pulled down by the 293mid-54, which was both interrupted by the mutation in the predicted target region of microDNA (Figure 5f). Taken together, these data indicate that SSLmicroDNAs likely interact with microRNAs in nuclei. The upper panel showed the sequence alignment we predicted between 293mid-28 and hsa-mir-1273g-3p, as well as 293mid-54 and hsa-mir-5096. The middle panel showed the mutant sequence region in 293mid-28 and 293mid-54. HEK293 cells were transfected with biotinylated 293mid-28 or negative control RNA (NC) or mutant 293mid-28 and then performed with the pulldown system based on interaction between biotin and streptavidin. The levels of miR-1273g-3p were detected by qRT-PCR in the pulldown product. n = 3, *p < 0.05 vs NC. The combination of 293mid-54 and miR-5096 was analyzed similarly to that of 293mid-28. (g) Analysis of SSLmicroDNA yield. The rates of SSLmicroDNA-positive clones relative to the total number of clones during purification were counted. *p < 0.05.

Discussion
Extrachromosomal DNAs exist in diversiform and are ubiquitously expressed in vivo, however, little is known about their function. Our present work identified a new form of extrachromosomal linear DNAs found in normal tissue and tumor cells. These DNAs were characterized by linear single strands, tens to hundreds of nucleotides length, a little higher than average GC content periodically intercepted by AT, TT, or AA dinucleotides. The single-stranded linear microDNAs were mainly localized at the non-coding regions of genome, where various SNP sites and strong H3K27Ac marks existed. SSLmicroDNAs, located in nuclei, were shown to interact with microRNAs in vivo. The difference in microDNAs content between normal tissues and tumor cells suggest that microDNAs may have a function in cellular defense against tumorigenesis. Briefly, our results revealed a new form of extrachromosomal DNAs, which could interact with microRNAs in the cell nucleus.
Circular microDNAs have been identified in normal tissues, and exhibit unique sequence features [7]. Compared to circular microDNAs, linear microDNAs own a similar length distribution and GC content; both forms shared AT, TT, or AA dinucleotide periodicity; circular microDNAs originate predominantly from functional regions such as exons, 5' UTRs, 3' UTRs, and CpG regions The upper panel showed the sequence alignment we predicted between 293mid-28 and hsa-mir-1273g-3p, as well as 293mid-54 and hsa-mir-5096. The middle panel showed the mutant sequence region in 293mid-28 and 293mid-54. HEK293 cells were transfected with biotinylated 293mid-28 or negative control RNA (NC) or mutant 293mid-28 and then performed with the pulldown system based on interaction between biotin and streptavidin. The levels of miR-1273g-3p were detected by qRT-PCR in the pulldown product. n = 3, * p < 0.05 vs. NC. The combination of 293mid-54 and miR-5096 was analyzed similarly to that of 293mid-28. (g) Analysis of SSLmicroDNA yield. The rates of SSLmicroDNA-positive clones relative to the total number of clones during purification were counted. * p < 0.05.

Discussion
Extrachromosomal DNAs exist in diversiform and are ubiquitously expressed in vivo, however, little is known about their function. Our present work identified a new form of extrachromosomal linear DNAs found in normal tissue and tumor cells. These DNAs were characterized by linear single strands, tens to hundreds of nucleotides length, a little higher than average GC content periodically intercepted by AT, TT, or AA dinucleotides. The single-stranded linear microDNAs were mainly localized at the non-coding regions of genome, where various SNP sites and strong H3K27Ac marks existed. SSLmicroDNAs, located in nuclei, were shown to interact with microRNAs in vivo. The difference in microDNAs content between normal tissues and tumor cells suggest that microDNAs may have a function in cellular defense against tumorigenesis. Briefly, our results revealed a new form of extrachromosomal DNAs, which could interact with microRNAs in the cell nucleus.
Circular microDNAs have been identified in normal tissues, and exhibit unique sequence features [7]. Compared to circular microDNAs, linear microDNAs own a similar length distribution and GC content; both forms shared AT, TT, or AA dinucleotide periodicity; circular microDNAs originate predominantly from functional regions such as exons, 5 UTRs, 3 UTRs, and CpG regions [7], whereas linear microDNAs are mainly produced from non-coding regions, such as intergenic or intragenic regions of genome.
Attention has been focused on extrachromosomal DNAs' general mechanisms of action, with no clear understanding as of yet. Viral DNA likely fails to integrate into host genomes and remains outside host chromosomes during viral infection, of which a typical example is human immunodeficiency virus (HIV) [36]. Additionally, DNA of the adeno-associated virus (AAV) exists mainly as circular episomes in human tonsil-adenoid, spleen, and lung tissues after infection [37]. Non-integrated Moloney murine leukemia virus (M-MuLV) DNA has been identified in a human rhabdomyosarcoma cell line (TE671 subline) [38]. Therefore, a bold hypothesis about the origin of linear microDNAs is that they could evolve from infectious organisms.
Cell defense mainly relies on the immune system [39]. DNA-protein crosslinking and non-coding RNAs have been shown to function directly in regulating cell defense [40,41]. Interestingly, HeLamicroDNAs demonstrated an irregular length distribution (Figure 2d-g) and lower yield during the extraction of SSLmicroDNAs (Figure 5g, and Table 1) from HeLa cells compared to normal tissues. This indicated that microDNAs may participate in maintaining the physiological environment against tumorigenesis. In summary, we identified a new form of extrachromosomal microDNAs and analyzed their characteristics. SSLMicroDNAs are located in the cell nucleus, and interacted with microRNAs. Our present work revealed a new kind of regulatory molecule that functions during physiological processes in cells, through targeting small non-coding RNAs.