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Self-priming DNA or what is self-priming?

What is self-priming?

Self-priming refers to the folding back of oligonucleotides. Self-priming allows amplification initiation without the need for an additional primer, a short, single-stranded DNA sequence used in PCR.

Oligonucleotides can form a stable secondary structure within themselves, such as developing a hairpin or a homodimer. Also, in some primer sets, the forward primer can interact with the reverse primer to form a heterodimer. Avoiding complementary sequences in primers or primer sets minimizes self-priming. Therefore, in most molecular biology experiments using PCR, self-priming is not desired. However, many viruses appear to utilize a self-priming mechanism.
 
Linear DNA virus genomes utilize self-priming for the initiation of DNA synthesis.

Bourguignon et al., in 1976, showed that the linear DNA of the non-defective parvovirus minute virus of mice (MVM) contains a stable hairpin duplex of approximately 130 base pairs located at the 5'-terminus of the genome. The research group used a combination of enzymatic and physical techniques for their study. MVM DNA is utilized as a template-primer by several DNA polymerases, including reverse transcriptases. The initiation of DNA synthesis in vitro occured within 100 bases of the 3'-end of the genome. This reaction utilizes the 3'-terminus of the viral DNA as a primer.

[ G J Bourguignon, P J Tattersall, D C Ward; DNA of minute virus of mice: self-priming, nonpermuted, single-stranded genome with a 5'-terminal hairpin duplex. Journal of Virology Oct 1976, 20 (1) 290-306. [PMC] ]

Salzman & Faisch, in 1979, reported that the 3’-prime terminal ends in the linear, single-stranded parvovirus genome can serve as self-priming DNA in vitro. The parvovirus genome contains double-stranded hairpin termini.

[ Salzman LA, Fabisch P.; Nucleotide sequence of the self-priming 3' terminus of the single-stranded DNA extracted from the parvovirus Kilham rat virus. J Virol. 1979 Jun;30(3):946-50. [PMC] ]

The self-priming hairpin structure allows the synthesis of double-stranded DNA fragments.

E. Uhlmann, in 1988, reported the synthesis of double-stranded DNA fragments from one long oligodeoxynucleotide. The method employes uses oligonucleotides with a short, inverted repeat at their 3′ end, forming a hairpin structure. The 3′ end of this hairpin serves as a primer in the Klenow (large) fragment of E. coli DNA polymerase I-mediated synthesis of the second DNA strand. Restriction enzymes allow removal of the loop structure. According to Uhlmann, this method for sequential cloning of gene fragments enables the synthesis of gene fragments of different size.

[ Uhlmann, E.; An alternative approach in gene synthesis: Use of long self-priming oligodeoxynucleotides for the construction of double-stranded DNA. 1988, Gene, 71, 1, 29-40. [Sciencedirect] ]

Mauriceville and Varkud plasmids are retroid elements that propagate in the mitochondria of some Neurospora crassa strains. Wang et al., in 1992, showed that the Mauriceville plasmid reverse transcriptase synthesizes full-length cDNA copies of in vitro transcripts beginning at the 3'-end with a preference for transcripts having the 3'-tRNA-like structure.

[ Wang H, Kennell JC, Kuiper MT, Sabourin JR, Saldanha R, Lambowitz AM. The Mauriceville plasmid of Neurospora crassa: characterization of a novel reverse transcriptase that begins cDNA synthesis at the 3' end of template RNA. Mol Cell Biol. 1992 Nov;12(11):5131-44. [PMC] ]

Retroviruses and retrotransposons depend on the reverse transcription of their messenger RNA into double-stranded DNA inserted into host cells' genomes. Levin, in 1996, reported that long terminal repeat (LTR)-containing viruses and transposons prime reverse transcription from tRNA molecules. However, non-LTR retro-elements utilize alternative mechanisms of priming. For example, the hepatitis B virus can prime minus-strand DNA synthesis with the hydroxyl group of a tyrosine residue near the N terminus of the reverse transcriptase. The Mauriceville plasmid replicates as a closed circular DNA in the mitochondria of some Neurospora strains. The plasmid initiates DNA synthesis without a primer. Recent work has suggested that a broad class of retroelements lacking LTRs prime their reverse transcription from nicks made in the target site DNA. Elements in this category include the yeast mitochondria DNA group II intron aI2 and the non-LTR retrotransposon R2Bm from Bombyx mori.

[ Levin, Henry L.; An Unusual Mechanism of Self-Primed Reverse Transcription Requires the RNase H Domain of Reverse Transcriptase To Cleave an RNA Duplex MOLECULAR AND CELLULAR BIOLOGY, Oct. 1996, p. 5645–5654. [PMC] ]

The interaction of HIV-1 genomic RNA and human tRNA(Lys)3 initiates viral reverse transcription. HIV RNA contains an adenosine-rich (A-rich) loop that mediates the complex formation between tRNA and viral RNA. A G-A pair and a U-turn motif stabilize the loop structure by stacking of the conserved adenosines. The stabilized loop is similar to the tRNA anticodon structure suggesting a possible role in reverse transcription initiation.

Figure 1: Structural models of the HIV-1 RNA A-rich hairpin loop ( PDB ID 1BVJ:Chain A, NGCGACGGTGTAAAAATCTCGCC )

[ Puglisi EV, Puglisi JD. HIV-1 A-rich RNA loop mimics the tRNA anticodon structure. Nat Struct Biol. 1998 Dec;5(12):1033-6. [Pubmed] ]

The presence of DNA hairpins at the ends of the poxvirus genome suggests a self-priming DNA replication model. The self-priming model suggests a rolling hairpin strand-displacement mechanism. A nick on one strand proximal to the hairpin by an unidentified nuclease generates a 3′ OH end, allowing the addition of deoxynucleotides. The strands fold back due to self-complementarity, and the replication complex continues adding deoxynucleotides to the distal hairpin and around it. The result is the formation of a concatemer. A reiteration of the process could lead to higher-order concatemers. The resolution of the concatemers by the Holliday junction (HJ) resolvase results in unit genomes.

[ Moss B.; Poxvirus DNA replication. Cold Spring Harb Perspect Biol. 2013 Sep 1;5(9):a010199.
doi: 10.1101/cshperspect.a010199. [PMC] ; Holliday Junction ]

Oligonucleotide strands containing phosphorothioate linkages are known to foldback and self-prime to allow amplification. In 2016, Jung and Ellington showed that templates containing optimized numbers of phosphorothioate linkages exhibit increased self-folding efficiency over an extended range of reaction temperatures.

The researchers adapted the self-folding mechanism for analytical applications by developing a variant termed phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP). The incorporation of phosphorothioate (PS) modifications into the DNA backbone reduced dsDNA's thermal stability and increased the self-folding of terminal hairpins. This method detects single nucleotide polymorphisms as well as non-nucleic acid analytes, such as alkaline phosphatase. The destabilizing of DNA duplexes by optimal incorporation of phosphorothioates helped repetitive refolding of self-priming amplicons over range of reaction temperatures from 54 °C to 66 °C.

[ Jung C, Ellington AD. A primerless molecular diagnostic: phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP). Anal Bioanal Chem. 2016 Dec;408(30):8583-8591. [PMC] ]

The self-priming synthesis of modified DNA is possible via extension of repeating unit duplex “oligoseeds”. Whitfield et al. incorporated the sterically‐demanding nucleotides 5‐Br‐dUTP, 7‐deaza‐7‐I‐dATP, 6‐S‐dGTP, 5‐I‐dCTP and 5‐(octadiynyl)‐dCTP into two extending oligoseeds ( [GATC]5/[GATC]5 and [A4G]4/[CT4]4 ) for the synthesis  of DNA over 500 bp long containing repeat sequences. The reported approach allows synthesis of DNA oligonucleotides with controlled numbers, position, and type of modification, and the overall length of the DNA. The result is a designed DNA containing sequence‐determined sites for chemical adaptations, targeted small molecule binding studies, or sensing and sequencing applications.

[ Colette J. Whitfield, Rachel C. Little, Kasid Khan, Kuniharu Ijiro, Bernard A. Connolly, Eimer M. Tuite, Andrew R. Pike; Self‐Priming Enzymatic Fabrication of Multiply Modified DNA. Chemistry. A European Journal. (2018 0 24, 57, 15267-15274. [Link] ]

More recently, in 2019, Park et al. designed a pool of self-priming replicators to select more efficient replicators. Ten random bases (R) were included in the original dumbbell-like structure to study self-priming oligonucleotides' replication mechanism.

[ Daechan Park, Andrew D Ellington, Cheulhee Jung, Selection of self-priming molecular replicators, Nucleic Acids Research, Volume 47, Issue 5, 18 March 2019, Pages 2169–2176. [PMC] ]

A self-priming hairpin-based isothermal amplification (SPHIA) enables nucleic acid detection. The method uses a hairpin probe (HP1) designed to open when binding to the target nucleic acid. Upon opening HP1, the self-priming domain within the HP1 stem region is exposed and rearranged to serve as a primer. The following extension reaction displaces the bound target nucleic acid. Recycled target nucleic acids open another HP1. Next, the extended HP1 continues with repeated extension and nicking reactions. The result is the production of a large number of triggers. The triggers enter and initiate the phase 2 reaction through binding to HP2 producing numerous target mimic strands (Target′). Target′ enters and activates the phase 1 reaction, which mimics the target nucleic acid. This approach makes many double-stranded DNA products (FPs). Duplex-specific fluorescent signaling allows the monitoring of the reaction response in real-time.

[ Ja Yeon Song, Yujin Jung, Seoyoung Lee, and Hyun Gyu Park; Self-Priming Hairpin-Utilized Isothermal Amplification Enabling Ultrasensitive Nucleic Acid Detection. Anal. Chem. 2020, 92, 15, 10350–10356. [ACS] ]

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