Antisense strand

Fig. 2 RNAi inducers used in antiviral strategies. In general, RNAi is induced either by transfection of synthetic siRNAs into cells, or by stable or transient intracellular expression of double-stranded siRNA precursors (shRNA, e-shRNA, IhRNA, or pri-miRNAs). After transcription in the nucleus shRNAs, IhRNAs and e-shRNAs are exported to the cytoplasm and subsequently diced into mature siRNAs. Pri-miRNAs modified to encode antiviral siRNAs first undergo cleavage by Drosha before they are exported to the cytoplasm. Here the antiviral pre-miRNAs (also called shRNA-miRs) are processed by Dicer into the mature miRNAs. After loading of the antisense strand of the siRNAs/miRNAs into RISC, the complex will target and cleave viral transcripts bearing the complementary sequences...

Fig. 6. Metal-crosslinking in antisense and antigene technology. The recognition of the target (sense) strand by the antisense strand is rapid and is followed by a slower irreversible metal crosslinking. 

Variations The poly(A) tailing kit (Ambion) produces a mRNA population with varying lengths of poly(A) tails, controlled by altering poly(A) polymerase concentrations and incubation times. An alternate method to incorporate a poly(A) tail is to clone a defined stretch of adenosines/ thymidines into the > UTR of the template pDNA. To allow transcripts to finish on an adenosine, the insert should be followed by a restriction site for an enzyme that cleaves 5 of the last antisense strand thymidine, such as Nsi I. In this way, the poly (A) tail can be incorporated directly into the... 

When you see a sequence written with only one strand shown, the 5 end is written on the left. Usually this sequence is also identical to that of the RNA that would be made from this piece of DNA when transcribed left to right. The DNA strand that has the same sequence (except U for T) as the RNA that is made from it is called the sense strand. The sense strand has the same sequence as the mRNA. The antisense strand serves as the template for RNA polymerase. 

RNA polymerase uses the antisense strand of DNA as a template. RNA is synthesized in the 5 to 3 direction. 

RNA polymerase makes a copy of the sense strand of the DNA using the antisense strand as a template (Fig. 5-8). The sequence of the primary transcript is the same as that of the sense strand of the DNA. RNA polymerase needs no primer—only a template. Either of the two DNA strands can serve as the template strand. Which DNA strand is used as the tern-... 

In addition to the known transcript (APOE SI) that translates into APOE, there are three additional transcripts in mice. Two of these transcripts, APOE S2 and APOE S3, which are predicted to be TM proteins, are transcribed from the sense strand. APOE SI is transcribed from the antisense strand and is complementary to exon 4 of APOE... 

Unlike the DNApolymerase reaction, RNApolymerases catalyze the transcription of only one of the two DNA strands. The two DNA strands are termed the sense strand and the antisense strand. It is the antisense strand that is transcribed by the RNA polymerases. Thus, the base sequence of the newly synthesized RNA strand is identical to the sense strand of the DNA template, except of conrse that U replaces T. 

F ig U re 1 3.1 A schematic view of RNA chain elongation catalyzed by an RNApolymerase. In the region being transcribed, the DNA double helix is unwound by about a turn to permit the DNAs sense strand to form a short segment of DNA-RNA hybrid double helix. That forms the transcription bubble. Note that the DNA bases in the bubble on the antisense strand are now exposed to the enzyme and are useable as a template for chain elongation. The RNApolymerase works its way down the DNA molecule until it encounters a stop signal. (Reproduced from D. Voet and J. G. Voet, Biochemistry, 3rd, edn, 2004 Donald and Judith G Voet. Reprinted with permission of John Wiley and Sons, Inc.)... 

Length of sense and antisense strands should be between 20 and 30 bp. 

General siRNA functionality Overall GC content 5 terminal GC content GC content is ideally between 30 and 65%. GC stretches of 9 or more nucleotides should be avoided The 5 terminal third of the antisense strand should not contain more than two GC base... 

How strong the overall GC content of an siRNA influences its activity remains controversial in the literature. While some studies claim that the optimal GC content of an siRNA is 30-50%, others have found that also GC-rich siRNAs with GC contents of about 60% are highly efficient (35, 36). We therefore recommend using siRNAs with an overall GC content between 30 and 65% of base pairing nucleotides. It has been shown that GC stretches of 9 or more nucleotides anywhere in the base pairing sequence of an siRNA reduce its efficiency. In addition, siRNAs with low GC content (not more than two GC base pairs) in the 5 terminal third of the guide (antisense) strand are likely to be potent siRNAs (36). Therefore these two parameters should be taken into account when designing siRNAs. 

Structural studies on Ago proteins revealed that the so-called MID domain binds the 5 end of the guide strand (39,40). Because of its central localization in the Ago protein, this domain has been named MID domain. Crystal co-structures and Kj measurements of the MID domain in combination with all four nucleotides at the 5 end revealed that uridine (U) binds with the highest affinity to the MID domain, adenosine (A) with a slightly reduced affinity, and cytosine (C) and guanosine (G) with more than tenfold less affinity (41). Therefore, siRNA guide (antisense) strands should ideally contain a U or A at the 5 end. C and G should be avoided. For the passenger (sense) strand 5 end, C and G should be selected in order to minimize strand incorporation (Fig. la). Based on our own unpublished data the nucleotide specificity is not only a tool to manipulate strand selection but siRNA strands with U or A at the 5 end show also a higher absolute affinity for Ago proteins and therefore are more likely to be potent siRNAs (see Notes 2 and 3). 

The PCR is a three-step cyclic process that repeatedly duplicates a specific DNA sequence, contained between two oligonucleotide sequences called primers (154,155). The two primers form the ends of the sequence of DNA to be amplified and are normally referred to as the forward and reverse primers. The forward primer is complementary to the sense strand of the DNA template and is extended 5 to 3 along the DNA by DNA polymerase enzyme (Fig. 27). The reverse primer is complementary to the antisense strand of the DNA template and is normally situated 200-500 base pairs downstream from the forward primer, although much longer sequences (up to 50 kbase) can now be amplified by PCR. The process employs a thermostable DNA polymerase enzyme (such as the Taq polymerase from Thermus aqualicus BM) extracted from bacteria found in hot water sources, such as thermal pools or deep-water vents. These enzymes are not destroyed by repeated incubation at 94 °C, the temperature at which all double stranded DNA denatures or melts to its two separate strands (155). 

Fig. 22.1. A model of gene silencing. Long, double-stranded RNAs (dsRNAs) are processed into 20-26 nucleotide small interfering RNAs (siRNAs) by Dicer (Step 1). The siRNAs associate with an RNA-induced silencing complex (RISC, Step 2), unwinding and activating in the process (Step 3). The antisense strand of the siRNA guides the RISC to complementary mRNA molecules (Step 4), which are cleaved and destroyed (Step 5). Sense and antisense RNA strands are indicated by thick and thin lines, respectively.

The antisense approach to pharmaceuticals is conceptually attractive and powerful. If a protein target and its sequence are known, then the sequence of the corresponding mRNA will also be largely known. If the exact mRNA sequence can be determined, then a complementary polynucleotide may be prepared to form a duplex. Longer complementary antisense strands give a more stable duplex. 

The term (XG + Xc) is the sum of the mole fractions of guanine and cytidine in the antisense strand. The mole fraction of any nucleobase is equal to the number of nucleotides containing that base divided by the total number of nucleotides in the oligonucleotide strand. [M+] is the molar concentration of monovalent cations. In a typical mammalian cell, [K + ] is 140 mM, and [Na+ ] is 10 mM. L is the length of the duplex in base pairs. Based on Equation 6.2 and the assumption that phosphorothioate oligonucleotides behave as regular DNA, fomivirsen (6.17) would have a predicted Tm of 59 °C. Equation 6.1 predicts 64 °C. More complex forms of Equation 6.2 with increased accuracy appear regularly in the literature.8... 

The mRNA is known as the sense strand because it is the portion of the DNA that is ultimately translated by the cell into proteins. An antisense strand is the other, complementary strand in DNAs double helix structure—or any nucleic acid that is complementary to, and can pair exactly with, at least part of a sense strand. 

Each RNA polymerase transcribes only one strand, the antisense (—) strand, of a double-stranded DNA template, directed by a promoter. Synthesis occurs 5 — 3 and does not require a primer. 

During transcription of information from DNA into mRNA, the two complimentary strands of the DNA partly uncoil. The sense strand separates from the antisense strand. The antisense strand of DNA is used as a template for transcribing enzymes that assemble mRNA (transcription), which, in the process produces a copy of the sense strand. Then, mRNA migrates into the cell, where other cellular structures called ribosomes read the encoded information, its mRNA s base sequence, and in so doing, string together amino acids to form a specific protein. This process is called translation. ... 

The sequence of nucleotides within the single-stranded mRNA is assembled according to the complementary-base-pairing (Chap. 7) instructions from one of the strands of duplex DNA, which contains the gene. The DNA strand that bears the same sequence as the mRNA (except for T instead of U) is called the coding strand or sense strand. The other strand of DNA which acts as the template for transcription is called the template or antisense strand. Some textbooks do not define sense" and antisense in the way described here, and for this reason it may be preferable to use coding and template when referring to a particular strand. 

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